CROSSTALK BETWEEN DEVELOPMENTAL AND TUMOUR- SPECIFIC SIGNALLING PATHWAYS: -RELATED SERINE PEPTIDASES AND NODAL IN PROSTATE CANCER

Mitchell G. Lawrence

Bachelor of Applied Science (Hons), QUT

Institute of Health and Biomedical Innovation School of Life Sciences, Queensland University of Technology, Brisbane, Queensland, Australia

Submitted in fulfilment of the requirements for the degree of Doctor of Philosophy

~2009~

KEYWORDS

Androgen Receptor (AR), β-catenin, Conditioned Matrix (CMTX), Cripto, Human Embryonic Stem Cell (hESC), Kallikrein-Related Serine Peptidase (KLK), Lefty, Microenvironment, Nodal, Prostate Cancer, Prostate-Specific Antigen (PSA), Wnt.

I ABSTRACT

Prostate cancer is an important male health issue. The strategies used to diagnose and treat prostate cancer underscore the cell and molecular interactions that promote disease progression. Prostate cancer is histologically defined by increasingly undifferentiated tumour cells and therapeutically targeted by androgen ablation. Even as the normal glandular architecture of the adult prostate is lost, prostate cancer cells remain dependent on the androgen receptor (AR) for growth and survival. This project focused on androgen-regulated expression, altered cellular differentiation, and the nexus between these two concepts.

The AR controls prostate development, homeostasis and cancer progression by regulating the expression of downstream . Kallikrein-related serine peptidases are prominent transcriptional targets of AR in the adult prostate. Kallikrein 3 (KLK3), which is commonly referred to as prostate-specific antigen, is the current serum biomarker for prostate cancer. Other are potential adjunct biomarkers. As secreted proteases, kallikreins act through cascades that may modulate the prostate cancer microenvironment. Both as a panel of biomarkers and cascade of proteases, the roles of kallikreins are interconnected. Yet the expression and regulation of different kallikreins in prostate cancer has not been compared. In this study, a spectrum of prostate cell lines was used to evaluate the expression profile of all 15 members of the kallikrein family. A cluster of genes was co-ordinately expressed in androgen- responsive cell lines. This group of kallikreins included KLK2, 3, 4 and 15, which are located adjacent to one another at the centromeric end of the kallikrein locus. KLK14 was also of interest, because it was ubiquitously expressed among the prostate cell lines. Immunohistochemistry showed that these 5 kallikreins are co-expressed in benign and malignant prostate tissue. The androgen-regulated expression of KLK2 and KLK3 is well-characterised, but has not been compared with other kallikreins. Therefore, KLK2, 3, 4, 14 and 15 expression were all measured in time course and dose response experiments with androgens, AR-antagonist treatments, hormone deprivation experiments and cells transfected with AR siRNA. Collectively, these experiments demonstrated that prostatic kallikreins are specifically and directly regulated by the AR. The data also revealed that kallikrein genes are differentially regulated by androgens; KLK2 and KLK3 were strongly up-regulated, KLK4 and KLK15 were modestly up-regulated, and KLK14 was repressed. Notably, KLK14 is located at the telomeric end of the kallikrein locus, far away from the centromeric cluster of kallikreins that are stimulated by androgens. These results show that the expression of KLK2, 3, 4, 14 and 15 is maintained in prostate cancer, but that these genes exhibit different responses to androgens. This makes the kallikrein locus an ideal model to investigate AR signalling.

II The increasingly dedifferentiated phenotype of aggressive prostate cancer cells is accompanied by the re-expression of signalling molecules that are usually expressed during embryogenesis and foetal tissue development. The Wnt pathway is one developmental cascade that is reactivated in prostate cancer. The canonical Wnt cascade regulates the intracellular levels of β- catenin, a potent transcriptional co-activator of T-cell factor (TCF) transcription factors. Notably, β-catenin can also bind to the AR and synergistically stimulate androgen-mediated gene expression. This is at the expense of typical Wnt/TCF target genes, because the AR:β-catenin and TCF:β-catenin interactions are mutually exclusive. The effect of β-catenin on kallikrein expression was examined to further investigate the role of β-catenin in prostate cancer. Stable knockdown of β-catenin in LNCaP prostate cancer cells attenuated the androgen-regulated expression of KLK2, 3, 4 and 15, but not KLK14. To test whether KLK14 is instead a TCF:β- catenin target gene, the endogenous levels of β-catenin were increased by inhibiting its degradation. Although KLK14 expression was up-regulated by these treatments, siRNA knockdown of β-catenin demonstrated that this effect was independent of β-catenin. These results show that β-catenin is required for maximal expression of KLK2, 3, 4 and 15, but not KLK14.

Developmental cells and tumour cells express a similar repertoire of signalling molecules, which means that these different cell types are responsive to one another. Previous reports have shown that stem cells and foetal tissues can reprogram aggressive cancer cells to less aggressive phenotypes by restoring the balance to developmental signalling pathways that are highly dysregulated in cancer. To investigate this phenomenon in prostate cancer, DU145 and PC-3 prostate cancer cells were cultured on matrices pre-conditioned with human embryonic stem cells (hESCs). Soft agar assays showed that prostate cancer cells exposed to hESC conditioned matrices had reduced clonogenicity compared with cells harvested from control matrices. A recent study demonstrated that this effect was partially due to hESC-derived Lefty, an antagonist of Nodal. A member of the transforming growth factor β (TGFβ) superfamily, Nodal regulates embryogenesis and is re-expressed in cancer. The role of Nodal in prostate cancer has not previously been reported. Therefore, the expression and function of the Nodal signalling pathway in prostate cancer was investigated. Western blots confirmed that Nodal is expressed in DU145 and PC-3 cells. Immunohistochemistry revealed greater expression of Nodal in malignant versus benign glands. Notably, the Nodal inhibitor, Lefty, was not expressed at the mRNA level in any prostate cell lines tested. The Nodal signalling pathway is functionally active in prostate cancer cells. Recombinant Nodal treatments triggered downstream phosphorylation of Smad2 in DU145 and LNCaP cells, and stably-transfected Nodal increased the clonogencity of LNCaP cells. Nodal was also found to modulate AR signalling. Nodal reduced the activity of an androgen-regulated KLK3 promoter construct in luciferase assays and attenuated the

III endogenous expression of AR target genes including prostatic kallikreins. These results demonstrate that Nodal is a novel example of a developmental signalling molecule that is re- expressed in prostate cancer and may have a functional role in prostate cancer progression.

In summary, this project clarifies the role of androgens and changing cellular differentiation in prostate cancer by characterising the expression and function of the downstream genes encoding kallikrein-related serine proteases and Nodal. Furthermore, this study emphasises the similarities between prostate cancer and early development, and the crosstalk between developmental signalling pathways and the AR axis. The outcomes of this project also affirm the utility of the kallikrein locus as a model system to monitor tumour progression and the phenotype of prostate cancer cells.

IV TABLE OF CONTENTS

Keywords ...... I Abstract...... II Table of Contents...... V List of Figures ...... IX List of Abbreviations ...... XI Statement of Original Authorship ...... XIV Publications...... XV Acknowledgments...... XVI 1 CHAPTER 1: INTRODUCTION AND LITERATURE REVIEW ...... 1 1.1 Introduction...... 2 1.2 Prostate Development...... 2 1.3 Androgens in Prostate Development ...... 3 1.4 Androgen Receptor Signalling...... 3 1.5 Androgens and Andromedins in Epithelial-Mesenchymal Interactions ...... 4 1.6 Cellular Differentiation in Normal Adult Prostate...... 5 1.7 Normal Prostate Function and Homeostasis ...... 6 1.8 Prostate Diseases...... 7 1.8.1 Benign Prostatic Hyperplasia ...... 7 1.8.2 Prostatic Intraepithelial Neoplasia...... 8 1.9 Prostate Adenocarcinoma ...... 8 1.10 Cell and Molecular Interactions in Prostate Cancer...... 9 1.10.1 Androgens and Prostate Cancer...... 9 1.10.2 Differentiation and Prostate Cancer...... 10 1.10.3 Developmental Signalling Pathways and Prostate Cancer ...... 11 1.10.4 Cancer as a Caricature of Development ...... 12 1.10.5 The Luminal Phenotype and Prostate Cancer...... 12 1.10.6 Prostate Specific Antigen/Kallikrein 3 and Prostate Cancer Diagnosis ...... 13 1.11 Kallikrein-Related Serine Peptidases...... 13 1.11.1 The Kallikrein-Related Serine Peptidase Locus ...... 13 1.11.2 Proteolytic Actions of Kallikreins ...... 15 1.11.3 Functional Roles of Kallikerins in Cancer Progression...... 15 1.11.4 Tissue-Specific Expression and Hormonal Regulation of Kallikrein-Related Serine Peptidases ...... 18 1.11.5 Expression of Kallikrein-Related Serine Peptidases in the Prostate...... 18 1.11.6 Hormonal Regulation of Kallikrein-Related Serine Peptidases...... 20 1.11.7 Kallikrein-Related Serine Peptidase Hormone Response Elements...... 21 1.11.8 Interaction Between Kallikrein-Related Serine Peptidases and Developmental Signalling Pathways...... 22 1.12 The Wnt Pathway ...... 22 1.12.1 Canonical Wnt Signalling...... 22 1.12.2 The Interaction Between β-catenin and the Androgen Receptor...... 25 1.12.3 The Wnt Pathway in Prostate Development, Homeostasis and Cancer...... 25 1.13 Exploiting the Convergence of Developmental and Cancer-Related Signalling ...... 28

V 1.14 The Nodal Pathway ...... 29 1.14.1 Activation and Regulation of the Nodal Axis ...... 29 1.14.2 The Role of Nodal in Embryonic Development and Cancer...... 32 1.15 Summary and Relevance to the Project ...... 34 2 CHAPTER 2: MATERIALS AND METHODS...... 37 2.1 Introduction ...... 38 2.2 General Reagent and Chemicals...... 38 2.3 Cell Lines...... 38 2.4 Cell Culture...... 39 2.5 RNA extraction...... 40 2.6 Reverse Transcription Polymerase Chain Reaction (RT-PCR) ...... 40 2.7 Quantitative RT-PCR (QRT-PCR) ...... 41 2.8 Ligation and Cloning of PCR Products ...... 41 2.9 Transformation and Purification of Constructs ...... 42 2.10 Extraction ...... 42 2.11 Western Blotting...... 42 2.12 Transfections ...... 43 3 CHAPTER 3: EXPRESSION AND REGULATION OF KALLIKREIN-RELATED SERINE PEPTIDASES IN PROSTATE CANCER...... 45 3.1 Introduction ...... 46 3.2 Materials and Methods ...... 48 3.2.1 Immunohistochemistry ...... 48 3.2.2 Analysis of Publicly Available Microarray Data...... 48 3.2.3 RT-PCR and QRT-PCR ...... 49 3.2.4 Clustering Kallikrein mRNA Expression in Prostate Cell Lines...... 49 3.2.5 Western Blotting...... 49 3.2.6 Steroid Hormone Treatments...... 49 3.2.7 Steroid Hormone Deprivation ...... 50 3.2.8 Collection and Concentration of Conditioned Media...... 50 3.2.9 Androgen Receptor Knock Down ...... 50 3.2.10 RNA Stability Assay ...... 51 3.3 Results ...... 52 3.3.1 Kallikrein 2, 3, 4, 14 and 15 are Co-expressed in a Subset of Prostate Cell Lines ...... 52 3.3.2 Kallikrein 2, 3, 4, 14 and 15 are Co-expressed in Benign and Malignant Glandular Epithelial Cells in Prostate Tissue...... 55 3.3.3 Prostatic Kallikreins are Differentially Regulated by Androgens in LNCaP and 22Rv1 Prostate Cancer Cells...... 60 3.3.4 Prostatic Kallikreins are Differentially Regulated by Hormone Deprivation...... 68 3.3.5 Androgen Receptor Knock Down Alters Prostatic Kallikrein Expression...... 72 3.3.6 Androgens Do Not Alter the mRNA Stability of Prostatic Kallikreins...... 75 3.3.7 Kallikrein Genes With Similar Expression Profiles are Clustered Together...... 77 3.4 Discussion...... 82 3.4.1 Kallikreins are Differentially Androgen Regulated and Expressed in Clusters ...... 82 3.4.2 Potential Mechanism of Kallikrein Transcriptional Regulation ...... 86 3.4.3 The Clinical Implications of Kallikrein Expression in Prostate Cancer...... 90 4 CHAPTER 4: THE ROLE OF Β-CATENIN IN KALLIKREIN-RELATED SERINE PEPTIDASE EXPRESSION...... 93 4.1 Introduction ...... 94

VI 4.2 Materials and Methods...... 96 4.2.1 Cell Culture ...... 96 4.3 Inhibition of GSK3β Activity ...... 96 4.3.1 Luciferase Assays...... 96 4.3.2 Androgen Treatments ...... 97 4.3.3 QRT-PCR ...... 97 4.3.4 β-catenin Knockout ...... 97 4.3.5 Protein Extraction and Western Blotting...... 98 4.4 Results...... 99 4.4.1 β-catenin is Required for Optimal Expression of Androgen-Regulated Kallikreins ...... 99 4.4.2 GSK3β Inhibition Increases KLK14 Expression...... 99 4.4.3 KLK14 Expression Does Not Correlate With E-cadherin Levels ...... 101 4.4.4 Induction of KLK14 expression with GSK3β Inhibitors is Independent of β-catenin 105 4.5 Discussion...... 107 5 CHAPTER 5: DEVELOPMENTAL SIGNALLING PATHWAYS IN PROSTATE CANCER: THE EXPRESSION AND FUNCTION OF THE NODAL AXIS...... 111 5.1 Introduction...... 112 5.2 Materials and Methods...... 115 5.2.1 Cell Culture ...... 115 5.2.2 Network Formation, DU145 Conditioned Matrices and Periodic Acid-Schiff Staining115 5.2.3 Human Embryonic Stem Cell Conditioned Matrices ...... 116 5.2.4 Soft Agar Assays ...... 116 5.2.5 Zymography ...... 116 5.2.6 RT-PCR and QRT-PCR...... 117 5.2.7 Immunohistochemistry ...... 117 5.2.8 Recombinant Nodal Treatment...... 118 5.2.9 Stable Transfections ...... 118 5.2.10 Luciferase Assays and R1881 Treatment ...... 118 5.2.11 Nodal and Kallikrein Co-transfections ...... 119 5.2.12 Recombinant Kallikrein Activation...... 119 5.2.13 E-Cadherin:Fc and Fibronectin Digests...... 119 5.2.14 Silver Staining ...... 120 5.2.15 Nodal Purification ...... 120 5.2.16 Pro-Nodal Digestions ...... 121 5.2.17 Western blots...... 121 5.3 Results...... 123 5.3.1 The Microenvironment Regulates the Plasticity of Prostate Cancer Cells ...... 123 5.3.2 Human Embryonic Stem Cell Conditioned Matrices Reduce the Aggressiveness of Prostate Cancer Cells...... 123 5.3.3 Human Embryonic Stem Cell Conditioned Matrices Decrease Nodal Expression ...... 126 5.3.4 The Nodal Axis is Expressed in Prostate Cancer Cells ...... 128 5.3.5 Nodal Expression in Prostate Tissue ...... 130 5.3.6 Prostate Cancer Cells are Responsive to Nodal...... 130 5.3.7 Nodal Increases the Clonogenicity of Prostate Cancer Cells...... 134 5.3.8 Nodal Antagonises Androgen Receptor Signalling...... 134 5.3.9 Cleavage of Nodal by Kallikrein-Related Serine Peptidases...... 137 5.4 Discussion...... 145 6 CHAPTER 6: GENERAL DISCUSSION...... 155 6.1.1 Androgens and Kallikrein Expression in Prostate Cancer...... 156 6.1.2 Exploiting the Convergence Between Prostate Cancer and Early Development...... 159 6.1.3 The Role of the Nodal Axis in Prostate Cancer...... 161 6.1.4 Interactions Between Androgen Receptor and Developmental Signalling Pathways ..162 6.1.5 Final Conclusions ...... 163 REFERENCES...... 165

VII APPENDICES...... 227 Appendix A: A Summary of Studies Examining Kallikrein Expression in Prostate Cancer...228 Appendix B: Steroid Hormone Regulation of Kallikrein-Related Serine Peptidases...... 231 Appendix C: Oligonucleotide Primer Sequences ...... 238 Appendix D: Primary Antibodies Used for Western Blots and Immunohistochemistry ...... 240 Appendix E: Expression of Kallikreins in Human Embryonic Stem Cells ...... 242

VIII LIST OF FIGURES

Figure 1.1. Organisation of the Kallikrein Locus and Structure Kallikrein Genes and ..14 Figure 1.2. The Expression of Kallikreins in a Range of Human Tissues...... 19 Figure 1.3. The Canonical Wnt Pathway ...... 24 Figure 1.4. The Nodal signalling Pathway...... 30 Figure 3.1. Comparison of Kallikrein Expression Levels in Prostate Cell Lines ...... 53 Figure 3.2. Expression of Kallikrein Proteins in Prostate Cell Lines...... 54 Figure 3.3. KLK3 and KLK15 Expression in Prostate Tissue ...... 56 Figure 3.4. KLK2 and KLK14 Expression in Prostate Tissue ...... 57 Figure 3.5. KLK4 Expression in Prostate Tissue...... 59 Figure 3.6. Kallikrein Immunohistochemistry with Pilot Tissue Microarrays ...... 61 Figure 3.7. Kallikrein Immunohistochemistry on Serial Sections of Prostate Tissue ...... 62 Figure 3.8. Kallikrein mRNA Expression in LNCaP Cells with a Time Course of R1881 Treatment...... 64 Figure 3.9. Dose Dependent Changes in Kallikrein Expression in R1881 Treated LNCaP Cells65 Figure 3.10. Kallikrein Protein Levels in LNCaP Cells Treated with R1881...... 67 Figure 3.11. Expression of Kallikrein 4 Variants in LNCaP Cells Treated with R1881...... 69 Figure 3.12. Changes in Kallikrein Expression in 22Rv1 Cells Treated with R1881...... 69 Figure 3.13. Kallikrein Expression in Hormone-Deprived LNCaP and 22Rv1 Cells...... 71 Figure 3.14. Bicalutamide Reverses R1881-Induced Changes in Kallikrein mRNA Levels...... 73 Figure 3.15. Expression of Kallikreins in LNCaP Cells with AR Knock Down...... 75 Figure 3.16. Kallikrein mRNA Stability in LNCaP Cells Treated with Ethanol or R1881...... 76 Figure 3.17. Androgen Regulation of Kallikrein Expression in LNCaP and 22Rv1 Cells Compared with Genomic Localisation...... 78 Figure 3.18. Expression of all 15 Kallikrein Genes in Prostate Cell Lines...... 79 Figure 3.19. Profiles of Phenotypic and Differentiation Markers in Prostate Cell Lines...... 81 Figure 4.1. Kallikrein Expression in β-catenin Knockout LNCaP Cells Treated with R1881. ..100 Figure 4.2. Changes in TCF Activity and Kallikrein Expression in DU145 and 22Rv1 Cells Treated with LiCl...... 102 Figure 4.3. Changes in Kallikrein 14 Expression Due to GSK3β Inhibition...... 103 Figure 4.4. KLK14 and KLK15 Expression in DU145 Sublines...... 104 Figure 4.5. Kallikrein 14 Expression in DU145 cells with β-catenin Knockdown...... 106 Figure 5.1. Vaculogenic Mimicry of DU145 Sublines...... 124 Figure 5.2. Human Embryonic Stem Cell-Conditioned Matrices Decrease the Aggressiveness of DU145 and PC-3 Cells...... 125 Figure 5.3. Decreased Nodal Expression in DU145 Cells Cultured on hESC CMTX ...... 127

IX Figure 5.4. Expression of the Nodal Axis in Prostate Cell Lines ...... 129 Figure 5.5. Nodal Expression in Prostate Tissue...... 131 Figure 5.6. Expression of Nodal in Adjacent Regions of Benign and Malignant Prostate...... 132 Figure 5.7. Nodal Stimulates Smad2 Phosphorylation in Prostate Cancer Cells...... 133 Figure 5.8. Nodal Increases LNCaP Clonogenicity ...... 135 Figure 5.9. Nodal Antagonises Androgen Receptor Activity...... 136 Figure 5.10. The Nodal Pro-Domain Cleavage Site and Expression..... 138 Figure 5.11. Transfected KLK4 Does Not Activate Nodal in Cos-1 Conditioned Media...... 139 Figure 5.12. Purification and Quantifaction of Pro-Nodal...... 141 Figure 5.13. KLK3, KLK4 and KLK14 Cleave Protein Substrates ...... 142 Figure 5.14. Cleavage of Pro-Nodal by Prostatic Kallikreins...... 144

X LIST OF ABBREVIATIONS

β-TrCP β-transducin repeat-containing protein µg microgram µM micromolar µL microlitre µm micrometer ABCG2 ATP-binding cassette transporter G2 ActRII activin receptor type II AF-2 activation function-2 AP-1 activator protein 1 AR androgen receptor ARE androgen response element APC adenomatous polyposis coli AVE anterior visceral endoderm BCA bicinchoninic acis BMP bone morphogenetic protein bp BPH benign prostatic hyperplasia BSA bovine serum albumin ºC degrees Celsius cDNA complementary DNA ChIP chromatin immunoprecipitation assay CIC cancer initiating cell CK1 casein kinase 1 CK cytokeratin cm centimeters CMTX conditioned matrix CSS charcoal-stripped foetal calf serum DAB 3,3’-diaminobenzidine DBD DNA-binding domain DHT 5α-dihydrotestosterone Dkk1 dickkopf 1 DMEM Dulbecco’s modified eagle’s medium DMSO dimethylsulphoxide DNA deoxynucleic acid dNTP deoxynucleotide triphosphate DTT dithiothreitol Dvl disheveled ECM extracellular matrix EDTA ethylene diamine tetra acetate EGF epidermal growth factor ELISA enzyme-linked immunosorbent assay ERα/β oestrogen receptor α/β ETOH ethanol EXE extraembryonic ectoderm Hrs hours FCS foetal calf serum FGF fibroblast growth factor Fzd frizzled GPI glycosyl-phosphatidylinositol GSK3β glycogen synthase kinase 3β

XI GR glucocorticoid receptor GST glutathione s- hESC human embryonic stem cell IGF insulin-like growth factor IGFBP insulin-like growth factor binding protein kb kilobases kDa kilodalton KLK kallikrein LB Luria Bertani LBD ligand-binding domain LCR locus control region LRP5/6 lipoprotein receptor-related protein 5/6 M molar MAPK mitogen-activated protein kinase min minutes mg milligram mL milliliter mM millimolar MMP matrix metalloprotease MMTV mouse mammary tumour virus mRNA messenger RNA ng nanogram nM nanomolar OD optical density PAR protease activated receptor PBS phosphate-buffered saline PCR polymerase chain reaction PI3K phosphoinositide 3-kinase PIN prostatic intraepithelial neoplasia pM picomolar PR progesterone receptor PSA prostate-specific antigen PSCA prostate stem cell antigen PTEN phosphatase and tensin homolog PTHrP parathyroid hormone-related protein QRT-PCR quantitative reverse transcription polymerase chain reaction RNA ribonucleic acid RO reverse osmosis rpm revolutions per minute RT room temperature RT-PCR reverse transcription polymerase chain reaction SDS sodium dodecyl sulphate SDS-PAGE sodium dodecyl sulphate polyacrylamide gel electrophoresis sec seconds SFRP2 secreted frizzled-related protein 2 shRNA short hairpin RNA siRNA short interfering RNA TAE Tris-acetate EDTA TBS-T Tris-buffered saline-Tween 20 TCF T-cell factor TGFβ transforming growth factor β Tfm testicular feminization TMA tissue microarray TNM tumour/node/metastasis U units

XII UGE urogenital epithelium UGM urogenital mesenchyme UGS urogenital sinus uPA -type UTR untranslated region UV ultra violet VEGF vascular endothelial growth factor Wif1 Wnt inhibitory factor 1

XIII STATEMENT OF ORIGINAL AUTHORSHIP

The work contained in this thesis has not been previously submitted to meet requirements for an award at this or any other higher education institution. To the best of my knowledge and belief, the thesis contains no material previously published or written by another person except where due reference is made.

Signature: ______Mitchell Lawrence

Date: ______

XIV PUBLICATIONS

Veveris-Lowe, T.L., Lawrence, M.G., Collard, R.L., Bui, L., Herington, A.C., Nicol, D.L., Clements, J.A. Kallikrein 4 (hK4) and prostate-specific antigen (PSA) are associated with the loss of E-cadherin and an epithelial-mesenchymal transition (EMT)-like effect in prostate cancer cells. Endocr Relat Cancer 2005;12(3):631-43.

Whitbread, A.K., Veveris-Lowe, T.L., Lawrence, M.G., Nicol, D.L., Clements, J.A. The role of kallikrein-related peptidases in prostate cancer: potential involvement in an epithelial to mesenchymal transition. Biol Chem 2006;387(6):707-14.

Hugo, H., Ackland, M.L., Blick, T., Lawrence, M.G., Clements, J.A., Williams, E.D., Thompson, E.W. Epithelial--mesenchymal and mesenchymal--epithelial transitions in carcinoma progression. J Cell Physiol 2007;213(2):374-83.

Lawrence, M.G., Veveris-Lowe, T.L., Whitbread, A.K., Nicol, D.L., Clements, J.A. Epithelial-mesenchymal transition in prostate cancer and the potential role of kallikrein serine proteases. Cells Tissues Organs 2007;185(1-3):111-5.

Lai, J., Myers, S.A., Lawrence, M.G., Odorico, D.M., Clements, J.A. Direct progesterone receptor and indirect androgen receptor interactions with the kallikrein-related peptidase 4 gene promoter in breast and prostate cancer. Mol Cancer Res 2009;7(1):129-41.

XV ACKNOWLEDGMENTS

I sincerely thank my principal supervisor, Professor Judith Clements, for mentoring me for so many years as a Dean’s scholar, Honours student, and finally PhD candidate. I also appreciate the help and support I received from my associate supervisor, Dr David Nicol. I am also grateful to the many past and present members of the Hormone Dependent Cancer Program for their assistance and advice.

Thank you to the Australian American Fulbright Foundation for giving me the opportunity to complete part of my studies in the United States, and to the Fulbright Commission staff for their guidance and support. I am also especially grateful to Professors Steven Balk and Mary Hendrix and their colleagues for generously hosting me in their laboratories.

Special thanks to Dr John Lai, Dr Naira Margaryan, Associate Professor Lynne-Marie Postovit, Ms Elizabeth Seftor, and Mr Carson Stephens for contributing experiments to this study and to Dr Hemamali Samaratunga, the Northwestern University Prostate SPORE and Australian Prostate Cancer Collaboration BioResource for providing patient specimens.

I also gratefully acknowledge the scholarships and travel bursaries I received from the Queensland University of Technology, School of Life Sciences, Institute of Health and Biomedical Innovation, International Proteolysis Society, and Cancer Council of Queensland, as well as a Growing the Smart State PhD grant from the Queensland Government.

Many thanks must also go to John Lai and Violet Mathieson for their help in editing this thesis.

Finally, I would like to thank Mum, Dad, Bek, and other family and friends for their ongoing patience, humour and support.

XVI

1Chapter 1: Introduction and Literature Review

1

1.1 Introduction Prostate cancer is a significant health issue for older men. According to the Australian Institute of Health and Welfare (AIHW; http://www.aihw.gov.au/cancer), prostate cancer is the most commonly diagnosed malignancy in Australian males apart from non-melanocytic skin cancer. The lifetime risk of prostate cancer is 1 in 5. Fortunately, there is a 100% 5-year survival rate for men diagnosed with localised prostate cancer (Jemal et al., 2008). This decreases to 32% in men with tumours that have invaded outside the prostate. Indeed, prostate cancer is the second leading cause of cancer-related deaths among Australian men after lung cancer (AIHW). A greater understanding of the factors that drive prostate cancer progression is required to develop improved biomarkers and therapeutic interventions. In this chapter, the cell and molecular basis of prostate development, normal physiology, and cancer are reviewed. Particular focus will be given to the molecules and signalling pathways that are further explored in this PhD project.

1.2 Prostate Development There are many recurring themes in prostate development, homeostasis, and tumour progression. The prostate is formed through dynamic interactions, guided by androgens, between cells and their microenvironment. In the tenth week of gestation, the human prostate begins to develop from outgrowths of the urogenital sinus (UGS) which is derived from embryonic endoderm (Lowsley 1912, Kellokumpu-Lehtinen et al., 1980). Most other male accessory sex glands arise from the Wolffian ducts, which are of mesodermal origin. The clusters of embryonic cells budding from the UGS, known as urogenital epithelium (UGE), elongate into the surrounding urogenital mesenchyme (UGM) as solid cords of cells (Hayward et al., 1996a, Hayward et al., 1996b). Like all other mesenchyme in the male reproductive tract, UGM is derived from mesoderm. After budding and elongation, UGE undergoes branching morphogenesis in response to inductive cues from the UGM to form a tree-like network of epithelial cells surrounded by mesenchymal cells (Prins and Putz 2008). Thus, UGM is essential for prostate development. This is emphasised by tissue recombinant experiments where UGM can direct the prostatic differentiation, not only of UGE, but of human embryonic stem cells, mouse and human prostate stem cells, urogenital and adult bladder epithelium, and female UGE (Cunha et al., 1983, Neubauer et al., 1983, Boutin et al., 1991, Donjacour and Cunha 1993, Taylor et al., 2006, Leong et al., 2008, Vander Griend et al., 2008). Indeed, isolated UGE does not form prostatic structures in the absence of UGM or when combined with embryonic skin mesenchyme (Cunha 1972b, Cunha 1972a). However, UGE does form prostate when recombined with seminal vesicle mesenchyme, suggesting some commitment towards prostatic differentiation (Donjacour and Cunha 1995). The interactions between UGE and UGM are reciprocal; just as UGM directs

2

UGE branching morphogenesis, UGE induces the differentiation of periductal UGM into smooth muscle cells, and interductal UGM into mature fibroblasts (Prins and Birch 1995, Hayward et al., 1996b). Finally, this complex pattern of epithelial and mesenchymal cells in the foetal prostate matures during puberty to form the intricate network of prostatic ducts and glands.

1.3 Androgens in Prostate Development All stages of prostate development require androgenic steroid hormones. In the human foetus, testosterone is produced by the testis after approximately eight weeks gestation and specifies the UGS to form prostatic tissue. In the developing prostate, testosterone is converted intracellularly by 5α-reductase to dihydrotestosterone (DHT), a more potent form of androgen (Wilson et al., 1981, Deslypere et al., 1992). The prostate does not develop in castrated rabbit or rodent embryos (Price 1936, Jost 1953, Cunha 1973, Lasnitzki and Mizuno 1977). Furthermore, UGS explants harvested before testis development and testosterone production fail to produce prostatic buds unless they are supplemented with androgens (Cunha 1973, Lasnitzki and Mizuno 1977). Androgens are not only necessary for normal prostate development in male embryos, but sufficient to induce prostate formation in genetically female embryos (Jost 1953, Takeda et al., 1986). The early pulse of androgens is critical for initiating prostate development. UGS retrieved after in vivo exposure to endogenous androgens still exhibits prostatic budding when cultured in the absence of androgens, albeit with lower efficiency (Cunha 1973, Lasnitzki and Mizuno 1977). Unlike the initiation of prostate development, subsequent branching morphogenesis is androgen-sensitive, but not androgen-dependent. Surgical or chemical castration of mouse embryos after branching morphogenesis has begun reduces the rate and extent of further branching, yet it does not completely inhibit this process (Donjacour and Cunha 1988). Human prostate branching morphogenesis is complete by the third trimester when testosterone production declines and remains low after birth (Wilson et al., 1981, Xia et al., 1990). The prostate remains quiescent until the sharp rise in androgens during puberty. The increase in androgens stimulates growth and maturation of the tracts of epithelial cells laid down during foetal development into ducts and luminal glands (Donjacour and Cunha 1988). The epithelial cells proliferate and those lining the acini differentiate and begin to secrete a range of prostatic proteins (Aumuller and Seitz 1990). Therefore initiation, branching morphogenesis, and maturation of the prostate are all distinct processes regulated by androgens.

1.4 Androgen Receptor Signalling The effects of androgens are mediated through the androgen receptor (AR), a ligand-dependent transcription factor that is a member of the nuclear receptor superfamily. AR is encoded by a single gene on the X and consists of four functional domains, the N-terminal

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transactivation domain, DNA-binding domain (DBD), hinge region, and C-terminal ligand- binding domain (LBD) (Chang and Kokontis 1988, Lubahn et al., 1988, Claessens et al., 2008). In the absence of androgens, heat shock proteins sequester the AR, which remains poised for ligand binding (Marivoet et al., 1992). Androgen binding to the LBD causes a conformational change in the AR that releases it from inhibitory heat shock proteins, promotes homodimerisation, and exposes a nuclear export signal in the hinge region (Heinlein and Chang 2002). Nuclear localised AR then binds via the DBD to androgen response elements (AREs) in the promoter and enhancer regions of target genes. Canonical AREs are palindromic hexameric repeats separated by a three nucleotide spacer ((A/G)G(A/T)ACA nnn TGTTCT) (Roche et al., 1992, Beato et al., 1995, Nelson et al., 1999a). Ligand-bound LBD interacts with the transactivation domain and both regions recruit coregulator proteins to facilitate chromatin remodelling and transcriptional initiation (Heinlein and Chang 2002, Schaufele et al., 2005). Since the AR is responsible for the biological actions of androgens, it is essential for prostate development. Male sexual organs fail to develop in humans with inactivating mutations of AR (Brown 1995).

1.5 Androgens and Andromedins in Epithelial-Mesenchymal Interactions Classical tissue recombination experiments have shown that AR in mesenchymal, rather than epithelial, cells is responsible for androgen-dependent prostate development. Different combinations of UGE and UGM from wild-type and Tfm (testicular feminisation) mice, which have a nonfunctional AR due to a frameshift mutation, were grafted and grown under the kidney capsule of recipient mice (Cunha and Lung 1978, He et al., 1991). In the presence of androgens, homotypic recombinants of wild-type UGE and UGM developed into prostate, unlike Tfm UGE and UGM grafts which formed vaginal structures. In heterotypic recombination experiments, wild-type UGE mixed with Tfm UGM also exhibited vagina-like differentiation, whereas Tfm UGE plus wild-type UGM developed into prostate. Therefore, epithelial AR is dispensable for all early stages of prostate development including bud formation, branching morphogenesis, epithelial proliferation, and canalisation of ducts. However, epithelial cells do require AR for correct secretory differentiation and final prostate maturation (Donjacour and Cunha 1993, Prins and Birch 1995, Simanainen et al., 2007, Wu et al., 2007).

The observations from tissue recombination studies suggest that specification, proliferation, and differentiation of prostate epithelial cells are primarily controlled by androgen-regulated paracrine factors from mesenchymal cells. Such factors are known as andromedins (Tenniswood 1986, Thomson 2001). Fibroblast growth factor 10 (FGF10) was one of the first proposed andromedins (Lu et al., 1999). It acts as a paracrine factor between mesenchymal and epithelial

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cells in many tissues, and loss of FGF10 results in agenesis of the prostate (Metzger and Krasnow 1999, Donjacour et al., 2003). FGF10 expression is stimulated by androgens in the UGS and in turn up-regulates developmental genes such as sonic hedgehog and Hoxb13 in epithelial cells (Pu et al., 2007). Correct prostate development requires a balance of stimulatory and inhibitory factors, suggesting that particular andromedins may also repress morphogenesis. In a microarray study that aimed to identify potential andromedins in mouse UGM, many candidates were inhibitors of the Wnt pathway (Pritchard and Nelson 2008). These included secreted frizzled-related protein 2 (SFRP2), SFRP4, dickkopf 1 (Dkk1) and Wnt inhibitory factor 1 (Wif1), all of which were androgen-regulated.

The expression and function of andromedins hints at the broader context of signalling pathways involved in prostate development. Many of these factors are highly conserved between species and between the prostate and other glandular structures. The basic “morphogenetic code” involves several multigene families such as Wnts, the transforming growth factor β (TGFβ) superfamily, FGFs, and Hedgehogs which form a network of autocrine, paracrine and juxtacrine signals between epithelial and mesenchymal cells (Hogan 1999). The spatial and temporal expression of these pathways is then regulated in an organ-specific manner to give rise to anatomically and functionally distinct tissues. In the developing prostate, androgens modulate the morphogentic pathways through mesenchymal AR. This confirms the importance of androgens in the prostate and demonstrates that AR and developmental signalling pathways are interconnected.

1.6 Cellular Differentiation in Normal Adult Prostate Differentiation of secretory epithelial cells is the last stage of prostate maturation during puberty. Adult prostate epithelium is composed of two stratified layers; luminal cells, which secrete seminal plasma proteins, and basal cells, which are separated from stromal cells by the basement membrane. In patient specimens, luminal and basal layers can readily be identified by their differing morphology. Normal luminal cells are tall columnar epithelial cells, whereas basal cells are small and cuboidal or flattened in appearance. They can also be distinguished by their expression of different cytokeratin (CK) proteins; luminal cells express CK8 and CK18, while basal cells express CK5 and CK14 (Sherwood et al., 1991, Yang et al., 1997).

There is a gradual turnover of cells in the normal adult prostate with new epithelial cells arising from a putative subpopulation of prostate stem cells (Taylor and Risbridger 2008). Definitive prostate epithelial stem cells that can reconstitute prostate glands from a single cell have so far been identified in mice but not humans (Leong et al., 2008). Nevertheless, a range of markers can be used to enrich the human prostate epithelial stem cell population. Human prostate stem

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cells express CD133, CD44, ATP-binding cassette transporter G2 (ABCG2), and high levels of α2β1 integrin, but do not produce AR, p63, prostate stem cell antigen (PSCA), or prostate- specific antigen (PSA/Kallikrein 3/KLK3) (Collins et al., 2001, Richardson et al., 2004, Huss et al., 2005). Although their niche is poorly defined, prostate epithelial stem cells are thought to reside in the basal compartment (Hudson et al., 2000). Through asymmetric division, prostate cancer cells may maintain their ability to self-renew and produce daughter transit amplifying cells with more limited proliferative capacity. Transit amplifying cells are also part of the basal layer, but express a different repertoire of proteins. They lack CD133 and ABCG2, but express the basal marker p63 (Tran et al., 2002, Garraway et al., 2003). Like prostate stem cells, transit amplifying cells also have high α2β1 integrin expression and no AR, PSCA or PSA (Uzgare et al., 2004). Transit amplifying cells mature into intermediate cells after a limited number of divisions. Intermediate cells express lower levels of p63, and up-regulate PSCA and AR mRNA, but not protein (Tran et al., 2002, Garraway et al., 2003). Both luminal and basal cytokeratins are expressed by intermediate cells (van Leenders et al., 2003). In the final stage of differentiation, intermediate cells migrate upwards into the luminal layer, cease expressing p63, PSCA and basal cytokeratins, and up-regulate AR and androgen-regulated genes such as PSA (Masai et al., 1990, Sherwood et al., 1991, Yang et al., 1997, Wang et al., 2001a). These luminal epithelial cells are terminally differentiated and do not proliferate (Bonkhoff et al., 1994).

Other cell types within the normal prostate also arise from resident tissue stem cells. In addition to basal and luminal epithelial cells, prostate epithelial stem cells differentiate into neuroendocrine cells (Rumpold et al., 2002). These rare single cells scattered throughout acini and ducts secrete a range of growth factors and express markers such as chromogranin A and synaptophysin, but not AR or PSA (Deftos et al., 1996, di Sant'Agnese and Cockett 1996). A distinct subpopulation of mesenchymal stem cells that gives rise to prostatic stroma has also been identified (Lin et al., 2007). These cells express stem cell antigens such as CD133 and can be directed to differentiate into other mesenchymal lineages, such as adipocytes and osteocytes in vitro.

1.7 Normal Prostate Function and Homeostasis Together with the seminal vesicles, prostatic luminal epithelial cells produce the major components of seminal plasma. Since some of the most rapidly evolving genes are involved in reproduction, there is a wide variation in the composition of seminal plasma between species (Mann 1974, Aumuller et al., 1990, Clark and Swanson 2005). Human seminal plasma contains high concentrations of zinc, prostaglandins, spermine, citric acid, and fructose, which may help regulate the pH of the microenvironment around spermatozoa, inhibit the female immune

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response to spermatozoa, and inhibit the growth of bacteria (Partin and Rodriguez 1998). The prostate also secretes large amounts of kallikrein serine proteases, including PSA (Veveris-Lowe et al., 2007). These proteases liquefy seminal plasma and enhance sperm motility by degrading the coagulum proteins semenogelin I and II (Christensson et al., 1990, Robert et al., 1997, Lovgren et al., 1999, de Lamirande 2007). Just as androgen signalling is essential for prostate development, it is necessary for the secretory activity of the adult prostate. Most of the factors that luminal epithelial cells secrete into seminal plasma are either transcriptionally regulated by AR or produced by androgen-regulated biosynthetic (Mann 1974, Montgomery et al., 1992).

Not only are androgens required for secretory activity but, also, for homeostasis of the normal adult prostate. The prostate regresses without androgens with apoptosis of epithelial cells and fibroblastic dedifferentiation of stromal cells (Sugimura et al., 1986, Hayward et al., 1996b). Castration-induced apoptosis of the normal prostate is due to a lack of AR signalling in stromal rather than epithelial cells (Kurita et al., 2001, Wang et al., 2001b). This indicates once again that epithelial and mesenchymal AR have different functions. Mesenchymal AR activity stimulates the production of proteins such as insulin-like growth factor 1 (IGF1), which has an anti-apoptotic effect on the epithelial cells (Ohlson et al., 2007). In comparison, androgen signalling in epithelial cells suppresses proliferation and induces terminal differentiation to the luminal phenotype (Berger et al., 2004). Epithelial-specific AR knockout after prostate development in transgenic mice causes hyperproliferation of basal epithelial cells that do not express luminal differentiation markers (Simanainen et al., 2007, Wu et al., 2007). Therefore, homeostasis of the normal prostate relies on a balance between the stimulatory and inhibitory actions of AR in mesenchymal and epithelial cells.

1.8 Prostate Diseases Benign and malignant diseases of the prostate are common causes of health complications amongst older men and arise when normal homeostasis is disrupted. The cellular and molecular interactions that guide prostate development and tissue renewal are also important in prostate diseases, but are often dysregulated and unbalanced. Benign prostatic hyperplasia (BPH), prostatic intraepithelial neoplasia (PIN), and prostate cancer, three of the most common prostatic diseases, are outlined below.

1.8.1 Benign Prostatic Hyperplasia The prostate is growth quiescent during early adulthood with a balance of proliferation and cell death (Litvinov et al., 2003). Over the age of 40, this equilibrium is gradually lost, leading to

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enlargement of the prostate and eventually BPH (Rhodes et al., 1999). Indeed, 70% of men have BPH by the age of 60 (Isaacs 1994). Since BPH usually occurs in the transitional zone of the prostate surrounding the urethra, it causes a variety of lower urinary tract symptoms (Untergasser et al., 2005). BPH is not a premalignant lesion because it involves excessive proliferation, but not neoplasia. Changes in the levels of steroid hormones may be one of the mechanisms that contribute to BPH. As DHT levels decline with age, the relative activity of oestrogen compared with androgen signalling increases, changing the growth kinetics of prostate cells (Zhang et al., 1997a, Shibata et al., 2000, Roberts et al., 2004). BPH is particularly associated with an increase in the number of stromal cells which stimulate neo-formation of ductal and acinar structures by epithelial cells (Shapiro et al., 1992, Untergasser et al., 2005). For this reason it has been proposed that BPH is due to a reawakening of the embryonic UGM-like inductive capacity of the adult prostatic stroma (McNeal 1978). Accordingly, there is altered expression of andromedins involved in branching morphogenesis and normal prostate homeostasis such as FGFs and members of the TGFβ superfamily (Untergasser et al., 2005). Therefore, when prostate homeostasis is disrupted, the signalling pathways that regulate prostate development facilitate prostate pathogenesis.

1.8.2 Prostatic Intraepithelial Neoplasia The earliest accepted stage of prostate carcinoma is high-grade PIN where malignant epithelial cells accumulate within architecturally benign glands without invading into the local stroma (McNeal 1969, McNeal and Bostwick 1986). PIN and cancer most commonly arise in the peripheral zone of the prostate which occupies the caudal end of the gland and surrounds the distal portion of the urethra (Qian and Bostwick 1995, Qian et al., 1997). Like BPH and prostate cancer, the prevalence of PIN increases with age, although it precedes the onset of carcinoma by more than 10 years (Sakr et al., 1993a, Sakr et al., 1993b, Qian et al., 1997). Consistent with PIN being a precancerous disease, its genotype and phenotype are midway between normal prostate epithelial cells and cancer (Bostwick and Qian 2004).

1.9 Prostate Adenocarcinoma At least 95% of prostate cancers are adenocarcinomas arising from the glandular epithelial cells (IARC, 2004). Suspected cases of prostate cancer are either detected directly, as palpable lumps during digital rectal examination or indirectly, as high or rising PSA titres in patient serum. Diagnosis is then confirmed by histological examination of multiple needle biopsies (Frydenberg and Wijesinha 2007). The aggressiveness of tumours is evaluated using the Gleason grading system (Gleason 1966, Gleason and Mellinger 1974). The two most prominent patterns are graded from 1 (most differentiated) to 5 (least differentiated) and added together to give the

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Gleason score, a powerful measure of patient prognosis (Albertsen et al., 1998). The pathological stage of prostate cancer is also determined using the TNM (tumour/node/metastasis) classification system that ranges from T1 tumours which are small, nonpalpable, and low grade, to T4 tumours that have invaded into adjacent organs.

Gleason score, TNM stage, serum PSA concentration, and patient preference, health, and life expectancy are all considered when deciding the most appropriate treatment options for prostate cancer. The most common treatments for localised prostate cancer are surgical removal of the prostate (radical prostatectomy) and radiation from an external source (external beam radiation therapy) or implanted radioactive pellets (brachytherapy) (Schostak et al., 2008, Sia et al., 2008). In men where the risk of treatment outweighs the potential benefits, tumours are simply monitored through watchful waiting (Wu et al., 2004). Since prostate cancer is initially androgen-dependent, hormone deprivation is another common treatment strategy, particularly for recurrent and metastatic disease (Huggins and Hodges 1941, Tammela 2004). There are various methods of hormone deprivation including surgical castration (orchiectomy) and chemical castration with drugs that inhibit systemic testosterone production, metabolism of andrenal androgens, or that act as direct AR antagonists (Tammela 2004). Different forms of hormone deprivation are often used simultaneously or in combination with other treatments such as radiation. Although 80-90% of tumours initially respond to androgen deprivation, within 12 to 33 months there is an inevitable relapse into castrate-resistant prostate cancer for which there are no curative treatments (Denis and Murphy 1993). Untreated locally invasive prostate cancer and castrate-resistant disease eventually metastasise to local sites such as the lumbar spine, pelvic lymph nodes and bladder, as well as to distant sites including the long bones, lungs and para- aortic lymph nodes (Saitoh et al., 1984, Bubendorf et al., 2000). Metastatic prostate cancer is fatal and most palliative treatment options focus on pain management (James et al., 2006).

1.10 Cell and Molecular Interactions in Prostate Cancer

1.10.1 Androgens and Prostate Cancer Prostate cancer arises as a result of genomic mutations and dysregulation of the pathways that control normal prostate homeostasis. Androgens are essential for specification, branching morphogenesis, maturation, and homeostasis of the prostate gland as well as for prostate cancer. In the normal prostate, androgens form the basis of the complex interactions between epithelial and mesenchymal cells. Stromal AR activity fosters the proliferation of epithelial cells via andromedins, whereas epithelial AR stimulates the differentiation of epithelial cells and expression of secretory proteins, but inhibits their proliferation (Cunha 1994). The epithelial cells in turn promote smooth muscle differentiation of the mesenchyme. Disruption of AR signalling

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in either the stroma or epithelial cells can cause tumourigenesis (Stanbrough et al., 2001, Wang et al., 2001b, Han et al., 2005). Furthermore, the effects of AR signalling become distorted in epithelial tumour cells. For example, over 50% of prostate cancers harbour translocations that create fusions between the promoters of androgen-regulated genes, such as TMPRSS2 and KLK2, and the coding regions of ETS transcription factors including ERG, ETV1 and ETV4 (Tomlins et al., 2005, Hermans et al., 2008, Kumar-Sinha et al., 2008). This means that the ETS genes, which stimulate in vitro proliferation and in vivo formation of high grade PIN, become androgen-regulated (Tomlins et al., 2005, Klezovitch et al., 2008). Therefore, in tumours with fusion genes, epithelial AR signalling may promote rather than inhibit proliferation. The other effect androgens have on normal prostate epithelial cells is to induce differentiation, at least partially through NKX3.1, an androgen-regulated homeobox transcription factor. NKX3.1 is commonly inactivated in prostate cancer though mutation, methylation, or deletion (Abate-Shen et al., 2008). Indeed, the NKX3.1 gene lies within the 8p21 chromosomal region that is deleted in up to 85% of prostate tumours (Bova et al., 1993, Carter et al., 1993, Swalwell et al., 2002). Loss of NKX3.1 in prostate cancer cells would alter their downstream responses to AR signalling. ETS fusion genes, inactivation of NKX3.1 and numerous other changes mean that the androgen-regulated growth of prostate epithelial cells becomes increasingly independent of stromal AR. This autonomy may eventually permit the growth of prostate cancer cells without prostatic mesenchyme as metastases.

Androgens are essential for all stages of prostate cancer progression. In castrate-resistant prostate cancer the lack of androgens is circumvented by further dysregulation of AR signalling. AR levels can increase through transcriptional up-regulation, stabilisation, and genomic amplification, while AR activity is enhanced by signalling pathways such as PI3K, protein kinase A and Raf/Ras/MAPK (Visakorpi 1999, Gregory et al., 2001a, Gregory et al., 2001b, Holzbeierlein et al., 2004). Intratumoural androgen levels increase with the up-regulation of enzymes that convert adrenal androgens to testosterone or synthesise androgens from cholesterol (Holzbeierlein et al., 2004, Stanbrough et al., 2006, Mostaghel et al., 2007, Locke et al., 2008, Montgomery et al., 2008). In addition, AR activation by weak androgens, other steroids, and previously inhibitory anti-androgens can be enhanced through genomic mutation, RNA editing, and alternative splicing of AR (Taplin et al., 1995, Tilley et al., 1996, Dehm et al., 2008, Martinez et al., 2008). Therefore, the AR pathway is co-opted by prostate cancer cells to maintain their growth and survival.

1.10.2 Differentiation and Prostate Cancer As noted with the Gleason grading system, prostate cancer progression is characterised by increasingly undifferentiated tumour cells. This implies that the differentiation program of

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epithelial cells is disrupted in prostate cancer. Just as normal prostate is renewed from a small number of stem cells, prostate cancer is thought to arise from a subpopulation of cancer initiating cells (CICs) with stem-cell like properties. Whether CICs are transformed prostate epithelial stem cells or dedifferentiated intermediate cells that have acquired self-renewal capability is a source of debate and there are data supporting both hypotheses (Collins et al., 2001, Gu et al., 2007, Miki et al., 2007, Vander Griend et al., 2008). Nevertheless, CICs likely become tumourigenic through multiple genomic mutations and dysregulation of their niche. Indeed, there is an expanded population of malignant precursor cells in prostate-specific PTEN and combined p53 and Rb knockout mice, (Wang et al., 2006a, Zhou et al., 2007). CICs divide and give rise to more differentiated progeny that constitute the bulk of the tumour including basal, luminal and neuroendocrine cells (Gu et al., 2007). However, the normal epithelial differentiation program is progressively altered in prostate cancer. There is a decrease in the proportion of basal cells which become so rare that loss of the basal layer is a hallmark of prostate cancer (Yang et al., 1999, Oliai et al., 2002). The luminal cells also become more basal-like; although they still express luminal markers such as CK18 and AR, there is a relative decrease in PSA expression. There is a concomitant increase in the expression of intermediate markers such as PSCA (Raff et al., 2008). Several factors may influence these changes. Deletion of tumour suppressor genes, activation of oncogenes that stimulate the cell cycle and increased growth factor signalling may permit otherwise quiescent luminal cells to proliferate. As mentioned, the AR axis is frequently co- opted by prostate cancer cells. Expression of AR in basal prostate cancer cells causes them to differentiate to a mixed luminal-basal phenotype with loss of the basal marker p63 and increased PSA expression (Berger et al., 2004). Therefore, the balance between cell death, tissue renewal, and cellular differentiation is lost in prostate cancer, partially driven by changes in AR signalling.

1.10.3 Developmental Signalling Pathways and Prostate Cancer As prostate cancer cells become increasingly undifferentiated, they begin to re-express molecules that regulate their developmental precursors. The morphogenetic code that regulates elongation and branching of UGE into UGM is invoked by epithelial tumour cells invading into surrounding stroma. For example, the gene expression profiles from PTEN knockout and c-myc over-expressing mouse models of prostate cancer bear a striking resemblance to changes in prostate development (Pritchard and Nelson 2008). Genes that were up-regulated, compared to normal littermates, were related to early prostate development, whereas genes that were down- regulated tended to be expressed in later stages of development. Similarly, another study has shown that the gene expression profile of budding mouse UGE is reactivated in the invasive stages of human prostate cancer (Schaeffer et al., 2008). The TGFβ superfamily, Wnt, FGF,

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Sonic Hedgehog, and Notch pathways are all important in both prostate development and prostate cancer progression (Karhadkar et al., 2004, Kwabi-Addo et al., 2004, Danielpour 2005, Verras and Sun 2006, Leong and Gao 2008, Prins and Putz 2008, Schaeffer et al., 2008). The downstream effects of all of these pathways are highly context dependent. Not only is the morphogenetic code interpreted in a prostate-specific manner during development but, also, during tumour progression. Indeed, there is no association between the gene expression profile of lung branching morphogenesis and prostate development or prostate cancer (Pritchard and Nelson 2008, Schaeffer et al., 2008). Therefore, poorly differentiated prostate cancer cells subvert the cellular and molecular interactions that maintain homeostasis and revert to the phenotype of their developmental precursors.

1.10.4 Cancer as a Caricature of Development It has long been noted that cancer progression mimics many aspects of normal development and tissue renewal (Bailey and Cushing 1925, Greaves 1986, Pierce and Speers 1988). Cancer has been described as a caricature of development because there is a “gross exaggeration” of some aspects and under representation of others (Pierce and Speers 1988). This concept is broadly applicable to many different malignancies including haematological, cutaneous, and soft tissue cancers as well as tumours in plants (Braun 1956, Pierce and Wallace 1971, Greaves et al., 1983, Al-Hajj et al., 2003, Quintana et al., 2008). As discussed, prostate cancer resembles, but does not recapitulate, prostate development. The focus of this project is to explore the similarities and differences between prostate cancer and development and how these can be exploited to reduce the aggressiveness of prostate cancer. The rest of this chapter is devoted to the particular molecules and signalling pathways that are the focus of this study.

1.10.5 The Luminal Phenotype and Prostate Cancer Prostate cancer cells become increasingly undifferentiated with tumour progression, yet they still maintain a partially luminal phenotype. This is an important difference between prostate cancer and development. Whereas secretory epithelial cells are abundant in prostate cancer, they are absent in the developing prostate until puberty. The androgen-dependent production of seminal plasma proteins is a defining characteristic of prostate luminal epithelial cells. These proteins are secreted into the glandular lumen in the normal prostate. However, in prostate cancer the breakdown of glandular architecture, disruption of the basement membrane and loss of apical- basal cellular polarity mean that seminal plasma proteins are secreted into the tumour microenvironment. These proteins then leak into the bloodstream and can be detected in the serum of prostate cancer patients.

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1.10.6 Prostate Specific Antigen/Kallikrein 3 and Prostate Cancer Diagnosis PSA is one of the most prominent secretory proteins produced by prostate luminal epithelial cells. The concentration of PSA in seminal plasma typically ranges from 0.3 to 3 mg/mL (Ahlgren et al., 1995). Very little PSA usually enters the bloodstream with levels of about 0.6 ng/mL (Kuriyama et al., 1980, Savblom et al., 2005). Leakage of PSA into the circulation markedly increases in prostate cancer (Kuriyama et al., 1980, Stamey et al., 1987, Lilja et al., 2008). Therefore, PSA is commonly used as a serum biomarker to monitor and detect prostate cancer. The traditional threshold of PSA for prostate cancer diagnosis is 4 ng/mL, although titres can increase to over 100 ng/mL with advanced prostate cancer (Lilja et al., 2008). The use of PSA as a diagnostic marker is particularly controversial because PSA is tissue rather than disease-specific (Stamey et al., 2004). BPH, which is prevalent in older men, also increases PSA levels (Kuriyama et al., 1980, Stamey et al., 1987). The PSA test is most valuable in younger men where BPH is less common. For men in their forties, small increases in PSA are highly predictive of the development of advanced prostate cancer in future decades (Ulmert et al., 2008). Several refinements of the PSA test help discriminate between BPH and prostate cancer. These include calculating the velocity and doubling time of increasing PSA concentrations over multiple tests (Carter et al., 1992), normalising PSA titres to the volume of the prostate (Benson et al., 1992), comparing the amount of free and complexed PSA (Christensson et al., 1993, Catalona et al., 1995) and measuring different cleavage products and isoforms of PSA (Mikolajczyk et al., 2000a, Mikolajczyk et al., 2000b, Nurmikko et al., 2001, Catalona et al., 2003). Widespread PSA testing has also led to stage migration where fewer cases of advanced and metastatic prostate cancer, compared with low grade and potentially clinically insignificant disease, are being detected (Ung et al., 2002). This means that better biomarkers are needed to help balance early diagnosis and over-diagnosis of prostate cancer.

1.11 Kallikrein-Related Serine Peptidases

1.11.1 The Kallikrein-Related Serine Peptidase Locus Some of the most promising adjunct biomarkers for prostate cancer are closely related to PSA. It is just 1 of 15 members of the kallikrein-related serine peptidase family, several of which are highly expressed in the prostate. PSA is also known as kallikrein 3 (KLK3) and will herein be referred to using this nomenclature. The human kallikrein locus spans 265 kilobases on 19q13.3- 13.4 and is the largest contiguous cluster of proteases in the (Figure 1.1 and Gan et al., 2000, Harvey et al., 2000, Yousef et al., 2000b). The kallikrein genes, KLK1-15, have highly conserved genomic structure with 5 coding exons and identical intron phasing, although there is a variable number of additional untranslated 5’ and 3’ exons (Clements et al., 2004). All

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Figure 1.1. Organisation of the Kallikrein Locus and Structure Kallikrein Genes and Proteins The relative location of genes (blue arrows) in the kallikrein locus is shown in the upper diagram. KLK31P is represented by a double-headed arrow. The arrows also indicate the direction of transcription. The typical structure of kallikrein genes is shown in the middle diagram (not drawn to scale). Coding regions are dark blue, while untranslated regions are light blue. There a 5 coding exons for all genes with additional non-coding exons (+/-) for some genes. The domains of the corresponding kallikrein protein are shown in the bottom diagram. The pre-domain, also known as the signal peptide, is required for secretion. Once the pro-domain is cleaved, the mature kallikrein protein has proteolytic activity. The approximate positions of the three conserved residues of the , His41, Asp96, and Ser189, are shown, as is the location of 183 which determines whether kallikrein have - or - like specificity.

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genes except KLK2 and KLK3 are transcribed from the negative strand in the telomere-to- centromere direction. In addition to the traditional kallikrein genes, the locus contains a kallikrein-like gene, KLKP1, which is rich in repetitive sequences and has a portion that is homologous to exon 2 of KLK1-3 (Yousef et al., 2004a, Lu et al., 2006, Kaushal et al., 2008). Several non-coding and one coding transcript, KRIP1, are transcribed from KLKP1.

1.11.2 Proteolytic Actions of Kallikreins All kallikrein genes encode serine proteases with the conserved catalytic triad of histidine, aspartate and serine residues. The predicted molecular weight of kallikreins range from 24-29 kDa, although they are often 5-8 kDa larger due to glycosylation. Kallikreins are synthesised as zymogens with 16-34 amino acid signal peptide for extracellular secretion and 3-37 amino acid pro-peptide that must be cleaved for enzyme activation. Some kallikreins autoactivate by cleaving their own pro-domain (KLK2, 5, 11, 12 and 14), all except KLK4 are activated by one or more other kallikreins and several (KLK6, 11, 12, 14) can be activated by different proteases such as , and uPA (Yoon et al., 2007, Yoon et al., 2008a, Yoon et al., 2008b). This means that kallikreins can act in enzyme cascades where the proteolytic response to a stimulus is amplified and modified depending on the particular proteases that are present. Once kallikreins are activated the catalytic triad mediates proteolysis, while other residues in the substrate binding pocket determine the specificity. Just like trypsin, most kallikreins (KLK1, 2, 4- 6, 8, 10-14) have an aspartate residue at position 183 (trypsin numbering). This gives themtrypsin-like specificity to preferentially cleve after and lysine residues (Lunwall and Bratsand 2008). KLK15 has similar specificity, although it has a glutamic acid at position 183. KLK3 has a serine, KLK7 an asparagine and KLK9 a glycine in this position which gives them chymotrypsin-like specificity to cleave after tyrosine residues. Although several kallikreins have similar substrate specificity, KLK1 is the only member of the family with true kallikrein activity, defined as the ability to cleave kininogens to produce vasoactive kinin peptides (Kraut et al., 1930, Werle and Fiedler 1969). Plasma kallikrein, or KLKB1, is also a kininogenase but is not a member of the kallikrein-related peptidase family, because it has a divergent gene structure and is located on a different chromosome (Asakai et al., 1987, Evans et al., 1988).

1.11.3 Functional Roles of Kallikerins in Cancer Progression In prostate cancer, leakage of kallikreins from disorganised glands not only makes them potential biomarkers but, as proteases, functional modulators of the tumour microenvironment. Although the proportion of mature enzyme has not been measured in vivo for most kallikreins, 89% of KLK3 in the prostate cancer microenvironment was shown to be proteolytically active using KLK3-specific fluorescent peptide substrates (Denmeade et al., 2001). There are many putative

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kallikrein substrates, yet the majority have only been examined with in vitro biochemical assays so their proteolysis needs to be confirmed with cell culture or in vivo experiments. The most common substrates are extracellular matrix proteins, growth factors, and cellular adhesion molecules. Accordingly, the normal physiological roles of the kallikreins include degradation of enamel matrix proteins during tooth maturation for KLK4 (Hu et al., 2007) and cleavage of desmosomal adhesion proteins during skin desquamation by KLK5 and KLK7 (Caubet et al., 2004). In the context of cancer, matrix remodelling and cleavage of cell adhesion proteins like E- cadherin by kallikreins may promote the invasion of tumour cells. Activation of other proteases by kallikreins may also enhance invasion. KLK1 activates MMP2 and MMP9, and KLK2 and KLK4 activate urokinase-type plasminogen activator (uPA) (Tschesche et al., 1989, Takayama et al., 1997, Takayama et al., 2001b, Beaufort et al., 2006). KLK1, 3-9 and 13 all increase the in vitro invasiveness of various cancer cell lines through reconstituted ECM solutions like Matrigel or defined matrices of collagen II/III, laminin, fibronectin and vitronectin (Webber et al., 1995, Wolf et al., 2001, Ghosh et al., 2004, Ishii et al., 2004, Kapadia et al., 2004a, Prezas et al., 2006, Johnson et al., 2007, Shinoda et al., 2007). In addition, KLK6 has been shown to enhance the invasion of MCA3D keratinocytes through chicken chorioallantoic membranes (Klucky et al., 2007). Kallikreins may also facilitate the dissemination of tumour cells through protease activated receptors (PARs), a family of G protein-coupled receptors that initiate downstream signalling and cellular migration upon protease cleavage (Ramsay et al., 2008b). PARs are over- expressed in prostate cancer and are activated by KLK1, 2, 4, 5, 6 and 14 (Houle et al., 2005, Oikonomopoulou et al., 2006, Black et al., 2007, Mize et al., 2008, Ramsay et al., 2008a, Stefansson et al., 2008). These studies suggest that one of the major pathophysiological functions of the kallikreins is to promote the invasion and migration of tumour cells.

The functions of some putative substrates imply that kallikreins may also stimulate the proliferation of tumour cells. Insulin-like growth factor binding proteins (IGFBPs) are cleaved by KLK2-5, 11 and 14 (Cohen et al., 1992, Plymate et al., 1996a, Rehault et al., 2001, Koistinen et al., 2002, Matsumura et al., 2005, Michael et al., 2006, Borgono et al., 2007b, Rajapakse and Takahashi 2007, Sano et al., 2007). Cleaved IGFBPs have reduced affinity for IGF1, increasing the bioavailability of this mitogenic, anti-apoptotic growth factor in the tumour microenvironment (Fielder et al., 1994). Proteolysis of IGFBP3 by KLK3 increases the proliferation of prostate stromal fibroblasts (Sutkowski et al., 1999). In contrast, most kallikreins, including KLK3, either decrease or do not affect the in vitro proliferation of tumour cell lines (Denmeade et al., 2003a, Prezas et al., 2006, Shinoda et al., 2007, Veveris-Lowe et al., 2007). KLK4 and KLK6 are exceptions, increasing the proliferation of some cell lines, but not others (Prezas et al., 2006, Klokk et al., 2007, Klucky et al., 2007, Veveris-Lowe et al., 2007). Therefore, the biological significance of IGFBP degradation by kallikreins is unclear. These

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observations emphasise the need to confirm the relevance of kallikrein substrates identified in biochemical assays in a biological context.

In addition to invasion and proliferation, several other functions of kallikreins in cancer progression have been proposed, although many of the substrates involved have not been identified. KLK3 is the most extensively studied kallikrein in tumour progression. Along with KLK4, KLK3 induced epithelial to mesenchymal transition with loss of E-cadherin and increased migration in PC-3 prostate cancer cells (Veveris-Lowe et al., 2007). KLK3 also increases the proliferation and osteoblastic differentiation of bone cells (Killian et al., 1993, Yonou et al., 2001, Gygi et al., 2002, Goya et al., 2006, Nadiminty et al., 2006), perhaps in part by activating latent TGFβ2 and degrading parathyroid hormone-related protein (PTHrP) (Killian et al., 1993, Cramer et al., 1996, Dallas et al., 2005). This is significant because prostate cancer bone metastases are osteoblastic with net bone production, unlike most tumour types that cause bone loss (Guise et al., 2006).

KLK3 may also regulate oxygen balance in tumours. It inhibits in vitro endothelial migration and tube formation induced by basic FGF and vascular endothelial growth factor (VEGF) (Fortier et al., 1999, Fortier et al., 2000, Fortier et al., 2003, Koistinen et al., 2008). Although, these studies are yet to be replicated in vivo, if KLK3 attenuates angiogenesis, it may retard tumour growth. Yet reduced vascularisation may also lead to hypoxia which increases the aggressiveness of prostate cancer cells (Chan et al., 2007). KLK3 has also been shown to stimulate the production of reactive oxygen species in prostate cancer cells (Sun et al., 2001). Reactive oxygen species trigger many of the same signalling pathways as hypoxia which promote prostate cancer progression (Galanis et al., 2008).

The role of kallikreins in modulating the local immune response is noteworthy given the importance of inflammation in prostate cancer pathogenesis (De Marzo et al., 2007). KLK3 has been reported to have both immunosuppressive and pro-inflammatory functions. For example, high concentrations of KLK3 inhibit T-cell proliferation and dendritic cell maturation (Kennedy- Smith et al., 2002, Aalamian et al., 2003). Conversely, KLK3 stimulates peripheral blood monocytes to produce interleukin 1α and β which trigger pro-inflamatory interferon γ secretion by natural killer cells (Kodak et al., 2006). Interestingly KLK7, which has chymotrypin-like substrate specificity like KLK3, activates pro-interleukin 1β (Nylander-Lundqvist and Egelrud 1997). Sequestration of proteolytically active kallikreins may indirectly alter the local immune response. Active KLK2-5 and 13 are all bound by the inhibitor, α2- macroglobulin, which is not only present in serum, but also expressed by prostate stromal cells (Christensson et al., 1993, Frenette et al., 1997, Kapadia et al., 2004b, Lin et al., 2005, Matsumura et al., 2005, Michael et al., 2006). Protease-bound α2-macroglobulin undergoes a

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conformational change that allows it to activate cell surface receptors and stimulate the proliferation of prostate cancer cells and macrophages (Misra et al., 2002, Misra et al., 2005). Cumulatively, these studies imply that kallikreins are not just passive biomarkers but functional mediators of many aspects of cancer progression.

1.11.4 Tissue-Specific Expression and Hormonal Regulation of Kallikrein-Related Serine Peptidases Serine proteases like trypsin and thrombin are centrally produced and systemically active. In contrast, most kallikreins are locally produced and locally active, except when they are secreted into bodily fluids like seminal plasma and breast milk (Shaw and Diamandis 2007). The tissue- specific roles of kallikreins are exaggerated in cancer progression as they leak from poorly formed glands into the tumour microenvironment. Different combinations of kallikreins are synthesised in each tissue. Given that kallikreins activate one another in enzyme cascades, their functions depend on the spatial and temporal expression pattern of other members of the kallikrein family. Figure 1.2 summarises the varied expression profile of the kallikrein locus in a range of normal and malignant adult and foetal tissues. Some kallikreins are expressed in a narrow range of tissues, especially KLK2 and KLK3, which are highly expressed in the prostate. Similarly, KLK6 is predominantly expressed in neuronal tissues. However, these narrowly expressed kallikreins are not completely tissue-specific. For example, KLK3 has been detected in salivary glands, brain and breast, albeit at much lower levels than the prostate (Olsson et al., 2005, Narita et al., 2006, Shaw and Diamandis 2007, Stone et al., 2009). KLK5 and 7-15 have a broader expression profile and are produced by a range of visceral organs that are rich in epithelial cells, including the prostate. Consistent with kallikreins being secreted proteases, immunohistochemistry has shown that they are primarily expressed in glandular epithelial cells (Petraki et al., 2006).

1.11.5 Expression of Kallikrein-Related Serine Peptidases in the Prostate All kallikreins have been detected in prostate tissue or cell lines at the mRNA or protein level (Harvey et al., 2000, Shaw and Diamandis 2007). However, KLK2, 3, 4, 11, 14 and 15 are the most highly expressed. Immunohistochemistry and in situ hybridisation experiments have localised kallikrein expression to luminal epithelial cells (Darson et al., 1999, Hooper et al., 2001, Kapadia et al., 2003, Dong et al., 2005a, Memari et al., 2007, Rabien et al., 2008). Accordingly, many kallikreins are secreted into seminal plasma (Michael et al., 2006, Veveris- Lowe et al., 2007, Emami et al., 2008, Emami and Diamandis 2008). Given that the kallikreins are produced by luminal epithelial cells which do not differentiate until puberty, it is assumed that most kallikreins are not expressed in the developing prostate. Very little or no KLK3

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Figure 1.2. The Expression of Kallikreins in a Range of Human Tissues. This heatmap summarises relative kallikrein expression levels in 52 different human tissues and cell lines. The data was extracted from a previous study using Rosetta microarray chips which have oligonucleotide probes spanning multiple exon:exon boundaries for each transcript (Johnson et al., 2003). The kallikreins are listed in centromeric to telomeric locus order. The location of particular probes is noted. For example, KLK1-1 denotes the probe at the first exon boundary of KLK1. The expression values were median centred and clustered for tissue type, while keeping the order of the kallikreins constant. Relative expression values can be compared between tissues for each kallikrein probe, but not between kallikreins for each tissue.

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staining has been observed in foetal prostate specimens compared with adult tissues (Xia et al., 1990, Popek et al., 1991, Adams et al., 2002). Intriguingly, KLK6 is more highly expressed in the prostate during puberty than in adulthood (Dhanasekaran et al., 2005). Kallikreins are also differentially regulated in prostate cancer. As mentioned, there is an increase in serum KLK3 levels in prostate cancer (Lilja et al., 2008). There is also greater concentrations of KLK2, 11 and 14 in serum, but no change in KLK8 and 10, and decreased KLK5 and 6 (Diamandis et al., 2000, Haese et al., 2000, Borgono et al., 2003, Parekh et al., 2007, Sardana et al., 2007, Vickers et al., 2008). However, altered kallikrein levels in serum are attributable to the breakdown of glandular architecture and may not correlate with changes in expression at the cellular level. Indeed, there is a relative decrease in KLK3 expression in prostate cancer cells compared with benign cells and PIN (Qiu et al., 1990, Yang et al., 1992, Hakalahti et al., 1993, Grande et al., 2000, Sterbis et al., 2008). Lower KLK3 mRNA expression correlates with increased risk of recurrent prostate cancer (Sterbis et al., 2008). The expression of other kallikreins in prostate cancer tissue is summarised in Appendix A. It is important to note that most studies have small numbers of samples. There is disagreement about KLK2 levels, although the majority of studies have reported increased expression in prostate cancer (Darson et al., 1997, Darson et al., 1999, Becker et al., 2000, Magklara et al., 2000b, Siivola et al., 2000, Herrala et al., 2001). Most studies have also observed greater KLK4, 11, 14 and 15 expression in prostate cancer specimens. Although their tissue- and disease-specific expression patterns have been characterised, there is only a superficial understanding of the transcriptional regulation of most kallikreins.

1.11.6 Hormonal Regulation of Kallikrein-Related Serine Peptidases Steroid hormones regulate the expression of many kallikreins and determine their tissue expression profiles. A summary of changes in kallikrein levels in response to steroid hormones is presented in Appendix B. Most kallikreins are up-regulated by androgens, oestrogens or progestins. Notably, androgens have been shown to increase the expression of every kallikrein gene in at least one system. Glucocorticoids and mineralocorticoids have more variable effects on kallikrein expression where genes are up-regulated, down-regulated or unchanged depending on the cell line.

Although, there is an extensive amount of data on steroid hormone regulation of kallikrein expression, apart from studies on KLK2 and KLK3, much of it is not definitive. Most studies use time points of 24 hours or more so it is not clear whether changes in kallikrein expression are direct or secondary effects of steroid hormones. Several studies used ELISAs to measure changes in kallikrein levels in conditioned media of cells cultured with or without hormones (Borgono et al., 2003, Luo et al., 2003a, Yousef et al., 2003c, Paliouras and Diamandis 2007, Shaw and Diamandis 2008). This technique requires 7 day treatments so that detectable levels of

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kallikreins accumulate in the media. However, it becomes difficult to determine whether changes in kallikrein levels are due to the direct action of steroid hormone receptors or changes in cellular proliferation and phenotype. Another shortcoming of many studies is that changes in kallikrein expression are not compared to well characterised hormone-regulated genes or other members of the kallikrein family. This makes it hard to judge how potently particular kallikreins are regulated. Furthermore, results are often ambiguous because in some cell lines steroid hormones may bind to other hormone receptors in addition to their cognate receptor. For example, KLK15 expression is stimulated by androgens, oestrogens, progestins and glucocorticoids in LNCaP cells in which the AR is mutated and can bind all of these ligands (Schuurmans et al., 1988, Yousef et al., 2001c, Shaw and Diamandis 2008). Only a few studies have co-treated cells with a hormones and a specific receptor antagonist to confirm which receptor the hormone is acting through (Luo et al., 2000, Luo et al., 2003a, Kulasingam and Diamandis 2007, Paliouras and Diamandis 2007, Kaushal et al., 2008). More detailed studies are required to confirm many of the preliminary reports of hormone-regulated kallikrein expression.

1.11.7 Kallikrein-Related Serine Peptidase Hormone Response Elements Direct hormone receptor binding sites have only been functionally characterised for the KLK2, KLK3 and KLK4 promoters. The AR is recruited to three regions within the KLK3 promoter. AREI is a high affinity proximal promoter ARE located 156 bp upstream of the transcription initiation site that closely conforms to the consensus ARE sequence (Riegman et al., 1991). AREI is sufficient to stimulate moderate levels of androgen-responsive gene expression in reporter assays. In contrast, AREII is a low affinity AR at -379 bp that may co- operate with other AREs to further enhance transcription (Cleutjens et al., 1996). The KLK3 enhancer region contains AREIII, which is a strong consensus ARE at -4134 bp from the transcription initiation site, surrounded by several low affinity AREs (AREIIIA, IIIB, IV, V and VI) (Schuur et al., 1996, Cleutjens et al., 1997, Zhang et al., 1997b, Farmer et al., 2001). This distal enhancer region is essential for maximal androgen-regulated gene expression. The KLK2 promoter is similar to KLK3. It has a proximal promoter ARE at -160 bp that resembles KLK3 AREI. KLK2 also has an upstream enhancer ARE at -3805 bp that is required for optimal androgen-mediated expression like KLK3 AREIII (Young et al., 1992, Murtha et al., 1993, Yu et al., 1999, Mitchell et al., 2000a). Recently, the KLK4 promoter was partially characterised. It contains a functional progesterone response element (PRE) that mediates modest levels of progesterone-responsive gene expression in breast cancer cells (Lai et al., 2009). The PRE lies 397 and 2419 bp upstream of the two KLK4 transcription initiation sites. A putative ARE was also identified, but it only interacts with AR indirectly and is insufficient to stimulate androgen

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regulated gene expression. This indicates that the KLK4 promoter is different from KLK2 and KLK3.

In summary, a significant difference between prostate development and tumour progression is the prevalence of luminal epithelial cells in prostate cancer. Kallikrein-related serine peptidases are abundantly produced by normal luminal epithelial cells and are maintained in prostate cancer, despite relative increases or decreases in the levels of particular kallikreins. As kallikreins leak from disorganised glands in prostate cancer they may become useful biomarkers and functional mediators of prostate cancer progression. Several kallikreins are highly expressed in the prostate, possibly due to androgen regulation. The androgenic control of KLK2 and KLK3 expression is well characterised, but the data is more preliminary for other kallikrein genes. Furthermore, little is known about the co-ordinated expression pattern of kallikreins in the prostate and their transcriptional regulation relative to one another. Therefore, a key objective of this project is to characterise the expression profile of the kallikrein locus in a spectrum of prostate cell lines and compare the transcriptional response of different kallikrein genes to androgens in prostate cancer.

1.11.8 Interaction Between Kallikrein-Related Serine Peptidases and Developmental Signalling Pathways. As a caricature of development, prostate cancer progression mirrors many aspects of tissue formation, although there are also distinct dissimilarities. One difference is the production of kallikreins by secretory luminal epithelial cells which are present in the normal and malignant prostate, but not during development. In prostate cancer cells, the signalling pathways that conform to development are integrated with those that differ. Accordingly, the expression of kallikreins is probably modulated by developmental factors that are re-expressed in prostate cancer. This is particularly likely because kallikreins are regulated by AR, which is central to the growth and survival of prostate cancer cells. The Wnt/β-catenin axis is one developmental pathway that may regulate kallikrein expression. The Wnt pathway is important in prostate morphogenesis, is reactivated in prostate cancer and converges with AR signalling.

1.12 The Wnt Pathway

1.12.1 Canonical Wnt Signalling The mammalian Wnt family is a large group of cysteine-rich, hydrophobic ligands that regulate a diverse range of biological process including embryonic induction and patterning, cellular polarity, specification of cell fate, and tissue morphogenesis (Clevers 2006, Nusse 2008). Wnts activate three different pathways; the canonical β-catenin/T-cell factor (TCF) cascade, the

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noncanonical planar cell polarity pathway, and calcium-dependent signalling (Katoh 2005, Kohn and Moon 2005). The β-catenin/TCF pathway is the most comprehensively characterised and is the focus of this project (see Figure 1.3). The purpose of the canonical pathway is to regulate the cytoplasmic levels of β-catenin, a multifunctional molecule that is also a component of cell membrane adherens junctions with E-cadherin (Brembeck et al., 2006). Membrane-bound β- catenin is highly stable, unlike the cytoplasmic pool of β-catenin, which is sequestered by a multiprotein destruction complex in the absence of Wnt signalling. This cytoplasmic complex is composed of the serine threonine kinases, glycogen synthase kinase 3β (GSK3β) and casein kinase 1 (CK1), as well as the tumour suppressor proteins, adenomatous polyposis coli (APC) and axin, which act as scaffolds (Kishida et al., 1998, Nakamura et al., 1998). GSK3β and CK1 sequentially phosphorylate β-catenin which is then recognised by β-transducin repeat-containing protein (β-TrCP), part of a dedicated E3 ubiquitin complex (Amit et al., 2002, Liu et al., 2002b, van Noort et al., 2002). Subsequently, ubiquitinated β-catenin is rapidly degraded by the proteasome (Aberle et al., 1997, Easwaran et al., 1999). The canonical Wnt pathway inhibits β- catenin degradation. The cascade is activated by a subset of Wnts which bind to frizzled (Fzd) transmembrane receptors and low density lipoprotein receptor-related protein (LRP) co- receptors, LRP5 and LRP6, at the cell surface (Bhanot et al., 1996, Hsieh et al., 1999). Fzds activate the dishevelled (Dvl) protein and LRP5/6 phosphorylate axin which, together, disrupt the β-catenin destruction complex and lead to increased levels of cytoplasmic β-catenin (Mao et al., 2001, Wharton 2003, He et al., 2004, Li and Bu 2005).

Stabilised β-catenin translocates to the nucleus and activates gene expression through the TCF family of transcription factors, of which TCF4 is most abundant in the prostate. In the absence of β-catenin, TCFs bind to a conserved motif (AGATCAAAGG) and inhibit transcription by binding to groucho/TLE co-repressor proteins (van de Wetering et al., 1997, Cavallo et al., 1998, Roose et al., 1998). β-catenin displaces the co-repressors from TCF and recruits a suite of co-activators that stimulate transcription (van de Wetering et al., 1997, Daniels and Weis 2005). Therefore, β-catenin and TCF form a bipartite transcriptional complex where β-catenin provides the transactivation functions and TCF contributes the DNA binding ability. Numerous β- catenin/TCF target genes have been identified and many are highly context-dependent. The proto-oncogene c-myc and cell cycle regulator Cyclin D1 were the first β-catenin/TCF-regulated genes to be identified in humans (He et al., 1998, Shtutman et al., 1999, Tetsu and McCormick 1999). Other prominent targets that are implicated in cancer progression include MMP3, 7 and26, VEGF and Met (Brabletz et al., 1999, Crawford et al., 1999, Zhang et al., 2001, Boon et al., 2002, Prieve and Moon 2003, Marchenko et al., 2004). The transcriptional targets of β- catenin and TCF in prostate cancer are largely unknown, although one candidate is the aryl hydrocarbon receptor which is involved in xenobiotic detoxification (Chesire et al., 2004).

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Figure 1.3. The Canonical Wnt Pathway In the absence of Wnt signalling, β-catenin is sequestered and phosphorylated by the multiprotein complex of axin, APC, CK1 and GSK3β. β-catenin is then ubiquinated by the complex containing β-TrCP and degraded by the proteosome. Without β-catenin, TCF represses target gene expression, whereas AR still stimulates transcription. When Wnts bind to the LRP5/6 and Fzd receptor complex, the β-catenin destruction complex disaggregates, leading to increased levels of β-catenin. Nuclear β-catenin activates TCF-mediated transcription and enhances the expression of AR target genes.

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1.12.2 The Interaction Between β-catenin and the Androgen Receptor In addition to TCF target genes, nuclear β-catenin may stimulate the expression of other genes in the prostate because it also binds to AR. The interaction between β-catenin and AR has been demonstrated with GST pull-down, yeast two-hybrid and mammalian two-hybrid assays, as well as co-localisation and co-immunoprecipitation experiments with prostate cancer cell lines and xenografts (Truica et al., 2000, Mulholland et al., 2002, Pawlowski et al., 2002, Yang et al., 2002, Song et al., 2003, Wang et al., 2008b). β-catenin only interacts with agonist-bound AR and, like other transcriptional co-activators, fails to bind in the presence of receptor antagonists such as bicalutamide and mifepristone (RU486) (Truica et al., 2000, Yang et al., 2002, Mulholland et al., 2003, Song et al., 2003, Masiello et al., 2004, Verras et al., 2004). Using a series of AR mutants, one study proposed that β-catenin binds to the activation function-2 (AF- 2) region of the AR LBD, which undergoes a conformational change in response to androgens (Song et al., 2003). However AF-2 mutations also abrogate ligand binding, so it is difficult to identify the specific residues that interact with β-catenin. The β-catenin motifs that interact with AR are better characterised. AR binds to armadillo repeats 5 and 6, the same region of β-catenin as TCF and E-cadherin (Hulsken et al., 1994, Pai et al., 1996, Huber and Weis 2001, Mulholland et al., 2002, Yang et al., 2002, Song et al., 2003, Song and Gelmann 2005). However, AR binds to distinct amino acids within this motif (Song and Gelmann 2005). Similar to its interaction with TCF, β-catenin acts as a co-activator of AR, recruiting other co-regulators and further stimulating androgen-regulated gene expression (Koh et al., 2002, Song et al., 2003, Li et al., 2004, Song and Gelmann 2005). The AR and TCF signalling pathways are mutually exclusive because both transcription factors bind to the same region of β-catenin, of which there is only a limited cellular pool. Androgen treatment and AR over-expression reduce TCF-mediated transcription, whereas TCF transfection inhibits AR activity (Chesire and Isaacs 2002, Pawlowski et al., 2002, Mulholland et al., 2003, Verras et al., 2004). The crosstalk between the canonical Wnt cascade and AR via β-catenin suggests that reactivation of this developmental pathway has important implications for prostate cancer progression.

1.12.3 The Wnt Pathway in Prostate Development, Homeostasis and Cancer The Wnt pathway has an important role in many stages of development and disease. In embryogenesis, the Wnt/β-catenin cascade is first activated upon fertilisation when sperm disrupts the polarity of oocytes (Tao et al., 2005). Later in development, Wnts are part of the morphogenetic code that regulates tissue formation (Hogan 1999, Clevers 2006). Wnt signalling is down-regulated in adulthood, but then re-awakened in cancer (Wang et al., 2008a). Wnts and Wnt inhibitors, such as SFRPs, Wifs and Dkks, have dynamic expression profiles in the foetal prostate (Joesting et al., 2005, Zhang et al., 2006, Prins and Putz 2008, Pritchard and Nelson

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2008, Schaeffer et al., 2008, Wang et al., 2008a, Yu et al., 2008). Indeed, the Wnt pathway is the most highly androgen-regulated signalling cascade in early prostate development (Schaeffer et al., 2008). The balance of activators and antagonists determines the temporal and spatial distribution of cytoplasmic β-catenin levels and regulates branching morphogenesis of the UGE. The expression and activity of the Wnt axis declines as morphogenesis is completed (Prins and Putz 2008, Pritchard and Nelson 2008, Wang et al., 2008a).

After the high levels of developmental Wnt signalling decrease, the pathway remains essential for homeostasis of adult tissues. Wnts regulate the maintenance and expansion of progenitor cells and guide self-renewal of the intestine, hair follicle, breast and haematopoietic system (Korinek et al., 1998, Okamura et al., 1998, Alonso and Fuchs 2003, Li and Rosen 2005, van Es et al., 2005). TCF/β-catenin transcriptional activity has been localised to p63-positive basal cells, implying that Wnts have a similar function in the adult prostate (Wang et al., 2008a). Furthermore, stabilisation of cytoplasmic β-catenin through recombinant Wnt3a treatments or APC knockout leads to a greater number of p63-positive basal cells (Bruxvoort et al., 2007, Yu et al., 2008). After castration-induced regression of the prostate, there are higher levels of nuclear β-catenin staining and transcriptional activity during androgen-stimulated re-growth when most epithelial cells have a basal phenotype (Chesire et al., 2002, Wang et al., 2008b). This is consistent with β-catenin regulating the proliferation and differentiation of prostate progenitor cells during tissue renewal.

The Wnt/β-catenin pathway is amplified in prostate cancer. Similar to development, many Wnt ligands and inhibitors are differentially regulated (Horvath et al., 2004, Zhu et al., 2004, Joesting et al., 2005, Ohigashi et al., 2005, Zi et al., 2005, Hall et al., 2008). Several other changes may also affect β-catenin levels. Genomic mutations that make β-catenin resistant to GSK3β phosphorylation and, therefore, degradation have been identified in approximately 5% of prostate cancers (Voeller et al., 1998, Chesire et al., 2000, Gerstein et al., 2002, Yardy et al., 2008). Inactivating mutations of axin, APC and βTrCP, which all control β-catenin degradation, have been found at similarly low frequency (Voeller et al., 1998, Gerstein et al., 2002, Yardy et al., 2008). PTEN is more commonly inactivated in prostate cancer patients, and may lead to increased β-catenin levels through GSK3β inhibition via PI3K and Akt, although some studies dispute this point (Persad et al., 2001, Sharma et al., 2002a, Liao et al., 2004, Mazor et al., 2004, Salas et al., 2004, Wang et al., 2004, Mulholland et al., 2006). In addition E-cadherin, which sequesters β-catenin at the cell adhesion complex, is frequently down-regulated in prostate cancer (Mason et al., 2002). Although a direct cause and effect relationship between E-cadherin loss and increased nuclear β-catenin has not been established in vivo, high E-cadherin levels repress the AR:β-catenin interaction and reduce in vitro proliferation of prostate cancer cells

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independently of E-cadherin’s role in cell-cell contacts (Sasaki et al., 2000, Yang et al., 2002, Wong and Gumbiner 2003, Cronauer et al., 2005, Verras and Sun 2005, Syed et al., 2008). Collectively, these cancer-related changes may increase β-catenin levels and, by extension, TCF and AR signalling.

Several mouse models have shown that dysregulated β-catenin signalling in the prostate causes tumourigenesis. In these models, exon 3 of β-catenin, which encodes the GSK3β phosphorylation sites, is deleted using loxP-mediated recombination. This renders β-catenin resistant to degradation by the cytoplasmic destruction complex. Prostate epithelial cells are targeted with the Cre transgene under the control of androgen-responsive promoters including NKX3.1, probasin and the mouse mammary tumour virus (MMTV). Stabilisation of β-catenin led to squamous metaplasia, the proliferation and multi-layering of basal cells and high grade PIN (Gounari et al., 2002, Bierie et al., 2003, Yu et al., 2008, Pearson et al., 2009). Adenocarcinoma was also reported in one study; mice with exon 3-deleted β-catenin and K-ras over-expression developed invasive prostate cancer (Pearson et al., 2009). Prostate-specific knockout of APC, a component of the β-catenin destruction complex, also caused prostate cancer (Bruxvoort et al., 2007). Interestingly, FoxA2, a developmental forkhead transcription factor, was re-expressed in mice with mutant β-catenin (Yu et al., 2008). FoxA2 is usually expressed in the anterior primitive streak, definitive endoderm and budding prostate, but not the mature prostate (Sasaki et al., 2000, Mirosevich et al., 2005). Therefore, increased β-catenin levels may be a cause as well as an effect of increased developmental signalling in prostate cancer.

β-catenin is also implicated in later stages of prostate cancer progression, in particular castrate- resistant prostate cancer. There are greater levels of β-catenin in prostate cancer cell lines after long-term androgen deprivation and in castrate-resistant LNCaP sublines and tumours (de la Taille et al., 2003, Yang et al., 2005b, Wang et al., 2008b). In the LNCaP tumour model, the β- catenin:AR interaction was only detected in castrate-resistant specimens and not androgen- responsive or newly castrated tumours (Wang et al., 2008b). By recruiting co-activators, β- catenin may increase the sensitivity of AR to low concentrations of androgens. Notably, β- catenin also enhances the activation of mutant forms of AR by estradiol and weak androgens such as androstenedione (Truica et al., 2000). The importance of β-catenin in castrate-resistant growth has also been demonstrated with mouse models. After mice with exon 3-deleted β- catenin are castrated, the remaining prostate epithelial cells proliferate in the absence of androgens (Yu et al., 2008). There is negligible proliferation in wild-type animals. Similarly, prostate tumours continue to grow in APC knockout mice after castration, whereas the prostate regresses in wild-type mice and does not proliferate without androgens (Bruxvoort et al., 2007).

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These studies suggest that reactivation of Wnt/β-catenin signalling and its crosstalk with AR help maintain the survival of prostate cancer cells.

In summary, Wnt/β-catenin signalling, which regulates prostate morphogenesis and tissue renewal, intensifies in prostate cancer and has a functional role in carcinogenesis and tumour progression. β-catenin not only activates TCF-target genes, but up-regulates androgen-regulated genes through its interaction with AR. However, these functions are mutually exclusive. Epithelial AR is unnecessary for all stages of prostate development except secretory differentiation, but is essential throughout prostate cancer, including castrate-resistant disease. This implies that β-catenin predominantly transactivates TCF in epithelial cells during prostate development. It also suggests that the interaction between β-catenin and epithelial AR may be cancer-specific. Mesenchymal prostate cells are likely to strike a different balance between AR and TCF activity, possibly effecting the production of andromedins. Given that kallikrein- related serine proteases are prominent androgen-regulated genes, a goal of this project is to examine co-ordinated changes in their expression profile in response to increased levels of β-catenin.

1.13 Exploiting the Convergence of Developmental and Cancer-Related Signalling The commonalities between cancer and development mean that molecules that are initially characterised in one context are often important in the other. For example, the hedgehog pathway was first shown to regulate prostate branching morphogenesis, then later cancer progression and metastasis (Lamm et al., 2002, Freestone et al., 2003, Berman et al., 2004, Karhadkar et al., 2004). In comparison, the Wnt antagonist SFRP1 was originally found to be up-regulated in cancer-associated prostate fibroblasts with subsequent studies revealing a role in prostate development (Joesting et al., 2005, Joesting et al., 2008). The convergence of malignant and developmental signalling also means that cancer cells are responsive to embryonic cells and vice versa. However, signalling cascades that are strictly controlled during embryonic development and tissue morphogenesis are highly dysregulated in cancer progression. Accordingly, several studies have shown that embryonic microenvironments reduce the aggressiveness of tumour cells, presumably by restoring the balance of regulatory cues (DeCosse et al., 1973, Brinster 1974, Mintz and Illmensee 1975, Pierce and Speers 1988, Hayashi et al., 1990, Postovit et al., 2006, Postovit et al., 2008b). In contrast, depending on the ratio of each cell type, embryonic cells exposed to tumour cells differentiate, undergo altered patterns of morphogenesis, or do not change (Brinster 1974, Mintz and Illmensee 1975, Topczewska et al., 2006, Abbott et al., 2008). Recently, a technique was developed where tumour cells are exposed to embryonic cues by culturing them on three dimensional matrices pre-conditioned with human embryonic stem cells

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(hESCs) (Postovit et al., 2006, Postovit et al., 2008b). So far this model has been shown to reduce the aggressiveness of breast cancer and melanoma cell lines. Therefore, a goal of this project is to characterise the effect of hESC conditioned matrix (CMTX) on prostate cancer cells.

The bidirectional signalling between embryonic and tumour cells has been exploited to identify novel factors that are involved in cancer progression. Topczewska and colleagues (2006) used zebrafish embryos as biosensors of reactivated developmental signalling in cancer cells. They discovered that blastula stage embryos microinjected with highly aggressive C8161 melanoma cells developed abnormal head outgrowths or body axis duplications. Under these experimental conditions, the fluorescently labelled melanoma cells remained at the apex of the ectopic structures and did not form tumour-like masses. Poorly aggressive, isogenically matched C81-61 cells, did not alter the phenotype of the zebrafish embryos. These results suggested that the aggressive melanoma cells re-express a developmental factor capable of recruiting and instructing the host tissue. Further experiments identified this factor as Nodal, a potent embryonic morphogen belonging to the TGFβ superfamily. The role of Nodal in embryonic development is well characterised and this report joined a growing body of work implicating the Nodal axis in cancer progression (Strizzi et al., 2005).

1.14 The Nodal Pathway

1.14.1 Activation and Regulation of the Nodal Axis Nodals are a group of highly conserved TGFβ superfamily ligands that are essential for chordate development. Humans, mice and chickens have one Nodal ligand, whereas multiple Nodal-like proteins have been identified in amphibians (Xnr1, 2, 4, 5 and 6) and zebrafish (cyclops, squint and southpaw) (Schier 2003). Unlike other developmental pathways such as Wnt and Notch, Nodals are not present in Drosophila or Caenorhabditis elegans, implicating them in more sophisticated stages of vertebrate development (Schier 2003, Hayward et al., 2008). Nodal signalling overlaps with activin, another member of the TGFβ superfamily, such that they elicit similar phenotypes in gain of function experiments (Green 2002). Like activin, Nodal dimerises and activates the type I serine-threonine kinase receptor, Alk4 (also known as activin receptor IB, ActRIB), and type II receptors, ActIIA or ActIIB (Figure 1.4 and Reissmann et al., 2001, Yeo and Whitman 2001, Yan et al., 2002, Shi and Massague 2003). Nodal and activin B, but not activin A, can also signal through Alk7 (Reissmann et al., 2001, Tsuchida et al., 2004, Xu et al., 2004). However, Nodal predominantly activates Alk4 rather than Alk7 in embryonic development (Jornvall et al., 2004, Ho et al., 2006). Nodals are unique among the TGFβ superfamily because they require co-receptors from the EGF-CFC family to potentiate activin

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Figure 1.4. The Nodal signalling Pathway. Pro-Nodal is cleaved by proteases such as and PACE. Dimers of mature Nodal bind to the receptor complex of Alk4/7, ActRIIA/B and GPI-linked EGF-CFC proteins (Cripto or Cryptic). This stimulates phosphorylation of Smad2 or Smad3, which interact with Smad4, translocate the nucleus and activate gene expression by binding to transcription factors such as FoxHI and members of the Mixer family. Nodal signalling can be inhibited by Lefty A and Lefty B which bind directly to Nodal and EGF-CFC proteins.

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receptor signalling (Shen and Schier 2000). Humans have two EGF-CFC receptors, Cripto and Cryptic, which are anchored to the plasma membrane via glycosyl-phosphatidylinositol (GPI) linkages (Shen and Schier 2000). Nodal is unable to form a complex with activin receptors without Cripto or Cryptic, whereas the EGF-CFC proteins directly interact with ALK4/7 in the absence of Nodal (Reissmann et al., 2001, Yeo and Whitman 2001, Bianco et al., 2002, Sakuma et al., 2002, Yan et al., 2002). Cripto and Cryptic have similar biochemical activity in the Nodal cascade, but have distinct expression profiles (Shen and Schier 2000). Cripto also stimulates the PI3K/Akt, Src and ras/raf/MAPK pathways independent of Nodal (De Santis et al., 1997, Kannan et al., 1997, Ebert et al., 1999, Bianco et al., 2003). No such activity has been reported for Cryptic.

When Nodal binds to the Alk/ActRII/EGF-CFC receptor complex, it triggers the type I receptor to phosphorylate Smad2 or Smad3, which subsequently interact with the common mediator Smad, Smad4 (Kumar et al., 2001, Yeo and Whitman 2001). Activated Smads then translocate to the nucleus and activate transcription by binding to FoxHI, a winged helix transcription factor, or Mixer homeobox proteins, such as Mixl1 (Germain et al., 2000, Hart et al., 2002, Kunwar et al., 2003). Many transcriptional targets of Nodal are context-dependent, however several components of the Nodal axis are consistently up-regulated, including Nodal itself (Adachi et al., 1999, Norris and Robertson 1999, Vincent et al., 2004, Saijoh et al., 2005).

Nodal activity is regulated by several extracellular factors. Nodal is secreted from cells as a pro- protein that is able to diffuse large distances of up to 500 µm (Sakuma et al., 2002). However, to bind to the ActIIR/Alk/EGF-CFC complex, Nodal must first be activated through proteolytic cleavage of the pro-domain (Beck et al., 2002, Guzman-Ayala et al., 2004, Ben-Haim et al., 2006, Mesnard et al., 2006). Mature Nodal is then rapidly endocytosed and degraded (Le Good et al., 2005, Blanchet et al., 2008). This means that proteolytic cleavage ultimately regulates Nodal activity and stability. In the mouse embryo, two serine proteases from the subtilin-like proprotein convertase family, furin (PACE2) and PACE4, cleave an RQRR motif in the Nodal pro-region which conforms to the consensus RXRR proprotein convertase motif (Constam and Robertson 1999, Beck et al., 2002). Significantly, unlike some targets of proprotein convertases, Nodal is activated extracellularly. For example, Nodal expressed in the mouse epiblast is activated by furin and PACE4 from the extraembryonic ectoderm (Beck et al., 2002, Guzman- Ayala et al., 2004, Ben-Haim et al., 2006).

The intensity of Nodal signalling is also regulated by specific inhibitors, of which Lefty proteins are the best characterised. Leftys are divergent members of the TGFβ superfamily that act as monomers rather than dimers (Tabibzadeh and Hemmati-Brivanlou 2006). Humans express Lefty A and Lefty B and have a pseudogene, Lefty 3 (Kothapalli et al., 1997, Yashiro et al.,

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2000). Leftys abrogate Nodal signalling by binding directly to Nodal, Cripto and Cryptic and preventing activation of the activin receptors (Chen and Shen 2004, Cheng et al., 2004). Furthermore, Leftys are able to diffuse and inhibit Nodal signalling at a distance (Chen and Schier 2002). As transcriptional targets of the Nodal cascade, Leftys balance the activity of Nodal signalling through negative feedback (Meno et al., 1998, Saijoh et al., 2000, Yashiro et al., 2000, Meno et al., 2001). These interactions between Nodal and Leftys conform to the reaction-diffusion model of molecules that form a gradient of activity that can pattern a field of cells (Meinhardt and Gierer 2000). Nodal is also inhibited by Cerberus, a multifunctional antagonist that also binds BMPs (Piccolo et al., 1999). In addition, two membrane-bound inhibitors, Tomoregulin-1 and Nicalin, attenuate Nodal signalling (Harms and Chang 2003, Haffner et al., 2004). Collectively, the expression of these inhibitors ensures that Nodal signalling is tightly regulated.

1.14.2 The Role of Nodal in Embryonic Development and Cancer Nodal is crucial for several stages of embryogenesis and has a functional role in tumour progression when it is re-expressed in cancer. Nodal is already expressed at the blastocyst stage of mammalian development (Mesnard et al., 2006). Accordingly, Nodal is also secreted by hESCs, which are derived from the inner cell mass of the blastocyst (Besser 2004, James et al., 2005, Vallier et al., 2005). After implantation, Nodal expression is maintained in the epiblast, the portion of the embryo that gives rise to the foetus (Mesnard et al., 2006). Nodal signalling maintains pluripotency and inhibits precocious differentiation of both hESCs and mouse epiblasts (Brennan et al., 2001, James et al., 2005, Vallier et al., 2005, Mesnard et al., 2006).

Nodal stimulates endoderm and mesoderm formation and axis specification through reciprocal inductive interactions between different parts of the embryo. Nodal and Cripto are expressed in a proximal to distal gradient in the epiblast and stimulate the formation of visceral endoderm at the distal end of the embryo (Ding et al., 1998, Brennan et al., 2001, Norris et al., 2002). As visceral endoderm rotates proximally to become anterior visceral endoderm (AVE), it establishes the anterior-posterior axis (Ang and Constam 2004). The secretion of Nodal inhibitors, Lefty and Cerberus, by the AVE focuses Nodal activity to the posterior pole of the embryo and produces the primitive streak (Perea-Gomez et al., 2002). Nodal signalling in the epiblast is also regulated by the extraembryonic ectoderm (EXE), the part of the embryo that eventually develops into the chorion and foetal components of the placenta. Furin and PACE4 are produced by the EXE and activate pro-Nodal locally and also diffuse to the epiblast to activate Nodal at its source (Beck et al., 2002). In the EXE, mature Nodal triggers expression of BMP4, which diffuses back to the epiblast. BMP4 stimulates Wnt3 expression in the epiblast, which further up-regulates Nodal secretion (Morkel et al., 2003, Ben-Haim et al., 2006). In addition, Nodal increases its own

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expression and that of Cripto through a positive feedback loop in the epiblast (Brennan et al., 2001, Norris et al., 2002). Induction of mesendoderm by Nodal during gastrulation is dose- dependent. For example, high levels of Nodal activity are required for definitive endoderm formation in zebrafish embryos, whereas mesendoderm still develops at more modest levels (Schier et al., 1997, Thisse et al., 2000). Neural tissues partially develop from anterior ectoderm. Given that Nodal induces endoderm and mesoderm at the expense of ectoderm, inhibition of anterior Nodal activity by AVE-derived Lefty is critical for this process (Piccolo et al., 1999, Thisse et al., 2000).

After gastrulation, Nodal, Cryptic and Leftys have an asymmetric expression pattern that contributes to left-right axis specification. Nodal is initially up-regulated by the Notch pathway on the left side of the node (Krebs et al., 2003, Raya et al., 2003), an embryonic structure at the anterior tip of the primitive streak. Nodal then diffuses to the left lateral plate, guided by the movement of cilia, and stimulates expression of the Nodal axis through an auto-regulatory loop (Brennan et al., 2002, Nonaka et al., 2002, Saijoh et al., 2005). Nodal-induced Lefty A (Lefty 1 in the mouse) expression in the axial midline acts as a barrier to restrict Nodal signalling on the right side of the embryo, whereas Nodal-mediated Lefty B (Lefty 2 in the mouse) secretion in the left lateral plate mesoderm establishes a gradient of Nodal activity (Meno et al., 1998, Saijoh et al., 2000, Meno et al., 2001, Yamamoto et al., 2003). A prominent transcriptional target of Nodal on the left side of the embryo is Pitx2, a transcription factor that regulates asymmetric organogenesis (Logan et al., 1998, Yoshioka et al., 1998, Shiratori et al., 2001). Through Pitx2, Nodal determines the eventual placement of organs in relation to the midline. Nodal, Cryptic and Pitx2 knockdown in mice and Cryptic mutations in humans lead to defects such as randomised positioning of some organs like the stomach and isomerism of others, including the heart and lungs, which normally have a distinctive left and right structure (Gaio et al., 1999, Bamford et al., 2000, Shen and Schier 2000, Liu et al., 2001, Brennan et al., 2002).

Nodal expression declines after specification of the left-right axis and is absent in most normal adult tissues. However, the Nodal axis is reactivated in certain instances of tissue remodeling. For example, Lefty A is produced during menstruation and stimulates MMP activity (Kothapalli et al., 1997, Cornet et al., 2005). Nodal has also been implicated in development and regeneration of pancreatic islets (Zhang et al., 2008) and growth of the breast during lactation (Kenney et al., 2004). Components of the Nodal axis are also expressed in ovarian follicles and the testes and may regulate cell turnover (Tabibzadeh et al., 1997, Wang and Tsang 2007). Like other developmental signalling pathways, the Nodal cascade is over-expressed in cancer. To date, Nodal has been detected in melanoma and testicular, colon and breast cancer cell lines (Adkins et al., 2003, Topczewska et al., 2006). Cripto has been more widely studied and is over-

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expressed in numerous malignancies including those of the breast, lung, colon, ovary, endometrium, stomach, pancreas, bladder, testis and prostate (Saloman et al., 2000, Xing et al., 2004). Nodal signalling is functionally active in tumour cells and increases their invasiveness, clonogenicity and tumourigenicity though the Smad cascade (Topczewska et al., 2006, Postovit et al., 2008b). Little is known about the role of Nodal in prostate cancer. Therefore, the expression and function of the Nodal axis in prostate cancer will be characterised in this study.

1.15 Summary and Relevance to the Project There are many similarities between prostate development, tissue renewal and cancer progression. Each process is reliant on androgens and is regulated by a common set of signalling pathways. However, there are also distinct differences. Normal prostate epithelial cells depend on andromedins and stromal AR to proliferate, whereas prostate cancer cells co-opt the AR axis to proliferate independently. Aggressive prostate cancer cells also have an interconverted phenotype, expressing markers of both basal and luminal cells. The continued expression of luminal proteins despite overall dedifferentiation may be due to the expression of AR.

The secretion of seminal plasma proteins, such as KLK3, is a notable difference between prostate epithelial cells in development and cancer. Since luminal differentiation does not occur until puberty, KLK3 is not expressed in the foetal prostate. Several other members of the kallikrein family of serine proteases are also highly expressed by luminal prostate cells, possibly under the control of androgens. The breakdown of glandular architecture in prostate cancer leads to the leakage of kallikreins into the tumour microenvironment and bloodstream. This means that kallikreins may be important functional mediators of cancer progression as well as candidate serum biomarkers. However, the co-ordinated expression profile of the kallikrein locus in prostate cancer is poorly defined.

Prostate cancer cells integrate the signals from developmental and cancer-specific pathways. The intersection of the Wnt/β-catenin cascade with the AR axis is a prominent example. Wnts are part of the morphogenetic code that guides branching of the UGE and may also regulate tissue renewal in the normal prostate. The Wnt pathway is reactivated in prostate cancer and β-catenin may influence the expression of androgen-regulated genes by acting as an AR co-activator. As candidate androgen-regulated genes, the expression of kallikreins may be modulated by Wnt signalling in prostate cancer.

The re-expression of developmental factors makes tumour cells susceptible to regulatory cues usually only associated with embryonic cells. Not surprisingly, embryos have been used as

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biosensors to identify novel developmental pathways that are reactivated in cancer. Using this technique the Nodal axis was shown to be up-regulated in melanoma and breast cancer. Nodal is essential for embryogenesis and recent studies demonstrate that it is also important in cancer progression. However, the Nodal axis has not been investigated in prostate cancer.

This project will address each of these themes as well as the molecular interactions between them. The hypotheses of this study are:

1) Kallikreins are associated with the luminal epithelial-phenotype and therefore, co-ordinately regulated by AR.. 2) Developmental signalling pathways alter androgen-regulated kallikrein expression in prostate cancer cells. 3) Prostate cancer cells co-opt the Nodal signalling pathway, which increases their aggressiveness.

To investigate these hypotheses, the specific aims of this project are:

4) To characterise the expression profiles of kallikrein-related serine peptidases in prostate cancer and compare their transcriptional regulation by androgens. 5) To examine the interaction between the AR and Wnt/β-catenin axes and its effects on kallikrein expression. 6) To investigate the response of prostate cancer cells to an embryonic microenvironment. 7) To characterise the expression and function of the embryonic Nodal signalling pathway in prostate cancer and its interaction with kallikrein-related serine peptidases.

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2Chapter 2: Materials and Methods

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2.1 Introduction General cell and molecular biology techniques that were used throughout this project are described in this chapter. Other methods will be outlined in the particular chapters where they were used.

2.2 General Reagent and Chemicals Unless otherwise noted, all analytical grade reagents and chemical were from Ajax Chemicals (Melbourne, VIC, Australia), BDH Chemicals (Kilsyth, VIC, Australia) or Sigma Chemical Company (Castle Hill, NSW, Australia).

2.3 Cell Lines RWPE-1, RWPE-2, LNCaP, 22Rv1, MDA-PCa-2b, PC-3 and DU145 prostate cell lines were all obtained from the American Type Culture Collection (ATCC; Manassas, VA, USA). RWPE-1 cells were originally isolated from the peripheral zone of a non-neoplastic prostate and immortalized with HPV-18, while RWPE-2 cells are a clonal derivate RWPE-1 cells transformed with v-Ki-ras (Rhim et al., 1994). Only RWPE-2 cells form tumours in nude mice (Rhim et al., 1994). Under basal conditions these cell lines are composed of progenitor, transit amplifying and intermediate epithelial cells that express little or no AR or KLK3 (Bello et al., 1997, Litvinov et al., 2006). Prolonged treatment with mibolerone, a potent androgen, fosters the differentiation of some luminal cells that express both AR and KLK3 (Bello et al., 1997). LNCaP cells, established from a left supraclavicular lymph node metastasis, have androgen- dependent growth kinetics and exhibit androgen-responsive KLK3 expression (Horoszewicz et al., 1980, Horoszewicz et al., 1983). A mutation in the LBD of AR (T877A) in LNCaP cells facilitates its activation by non-androgenic steroid hormones and the AR-antagonist hydroxyflutamide (Veldscholte et al., 1990a, Veldscholte et al., 1990b, Taplin et al., 1995). 22Rv1 cells were isolated from a primary prostate cancer xenograft after castration induced regression and relapse in mice (Sramkoski et al., 1999). 22Rv1 cells therefore represent castrate- resistant but androgen-responsive prostate cancer. Like LNCaP cells, the AR LBD is mutated (H874Y) in 22Rv1 cells and has promiscuous affinity for other ligands (Tepper et al., 2002, Attardi et al., 2004). Furthermore, exon 3 of AR is duplicated leading to an expansion of the DBD (Tepper et al., 2002). 22Rv1 cells express two additional alternatively spliced forms of AR that lack the LBD and are constitutively active (Tepper et al., 2002, van Bokhoven et al., 2003b, Dehm et al., 2008). MDA-PCa-2b cells were generated from a paraspinal metastasis in a patient with castrate-resistant prostate cancer; however, they exhibit hormone-dependent growth and hormone-responsive gene expression as tumours in nude mice (Navone et al., 1997). The AR

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has the same T877A mutation as LNCaP cells as well as a L701H substitution, also in the LBD. This mutation facilitates AR activation by glucocorticoids and alters interactions between AR and chaperone proteins (Matias et al., 2002, Robzyk et al., 2007). PC-3 cells, derived from a lumbar vertebral metastasis (Kaighn et al., 1979), and DU145 cells from a brain metastasis (Stone et al., 1978), are both androgen-independent, androgen-insensitive and lack AR (Chlenski et al., 2001). Although some DU145 and PC-3 sublines expressing low levels of AR have been reported, the receptor does not stimulate androgen-regulated gene expression in these cases (Buchanan et al., 2004, Alimirah et al., 2006).

Several non-prostatic cell lines were also used in this study. COS-1 cells, a fibroblast-like cell line derived from African green monkey kidney (Gluzman, 1981), were used for transient and stable transfection experiments. Highly aggressive, C8161, and poorly invasive, C81-61, melanoma cells were used as controls for Nodal experiments. Both cell lines were derived from an abdominal wall metastasis of a patient with recurrent melanoma (Bregman and Meyskens, 1983, Welch et al., 1991, Hendrix et al., 1992). The H9 human embryonic stem cell line used in Chapter 5 was derived from the inner cell mass of a blastocyst stage human embryo and has a normal XX karyotype (Thomson et al., 1998) .

2.4 Cell Culture All cells were maintained at 37°C with 5% CO2 in an IR Sensor Incubator (Sanyo, Quantum Scientific, Brisbane, Australia). LNCaP, 22Rv1, DU145 and PC3 cells were cultured in RPMI 1640 medium (Invitrogen, Mount Waverly, VIC, Australia) containing 10% foetal calf serum (FCS, Invitrogen), 50 U/mL Penicillin G and 50 ug/mL Streptomycin (Invitrogen). RWPE-1 and RWPE-2 cells were grown in Keratinocyte serum-free medium with 50 µg/mL bovine pituitary extract and 5 ng/mL recombinant human epidermal growth factor (EGF) (Invitrogen). MDA- PCa-2B cells were maintained in BRFF-HPC1 media (AthenaES, Sapphire Biosciences, Sydney, Australia) supplemented with 20% FCS. The culture media was replaced every 2 to 4 days. The cells were passaged using 1x trypsin (Invitrogen) once they reached approximately 80% confluence. Cultures were tested for Mycoplasma infection each month using a Takara Bio PCR detection kit (Scientifix, Cheltenham, VIC, Australia).

For cryopreservation, cells were resuspended in culture media containing 10% dimethyl sulphoxide (DMSO, Sigma) and aliquoted into cryovials. The cells were frozen to -80 ºC at 1 ºC/min in a cryovessel filled with isopropanol and then transferred to liquid nitrogen for long term storage. To resuscitate stocks, cells were rapidly thawed at 37 ºC, resuspended in 20 mL of culture media and transferred into T80 cm2 flasks (Medos, Brisbane, QLD, Australia). The cells were allowed to seed down overnight before the culture media was replaced.

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To calculate cell numbers, cell suspensions were drawn up into NucleoCassettes and measured with a NuceoCounter (ChemoMetec, Allerof, Denmark). Alternatively, cell suspensions were mixed with 0.4% Trypan blue and manually counted using a haemocytometer (ProSciTech, Thuringowa, QLD, Australia).

2.5 RNA extraction Total RNA was extracted using TRIzol (Invitrogen) which was added directly to cells and incubated for 5 mins at room temperature. For example, 500 µL of TRIzol was added per well to 6-well plates. After the solution was transferred to RNase-free tubes, 200 µL of chloroform per mL of TRIzol was added and the mixture was shaken vigorously for 30 sec. The samples were incubated for 2-3 mins at room temperature and then centrifuged at 12 000 x g for 15 min at 4 ºC. The aqueous phase was transferred to a new tube and mixed with 500 µL of isopropanol per mL of TRIzol. RNA was precipitated for 30 min at room temperature or overnight at -20 ºC. The precipitate was pelleted by centrifuging samples at 14 000 x g for 15 min at 4 ºC. The supernatant was then discarded and the pellets washed with 1 mL of 70% ethanol per mL of TRIzol. The samples were centrifuged for another 10 min, the ethanol removed and the pellets allowed to air dry. The RNA was then resuspended in 20-50 µL of nuclease-free water (Invitrogen). RNA concentrations were measured using a NanoDrop 1000 spectrophotometer

(Thermo Scientific, Biolab, Scoresby, VIC, Australia) where an A260 optical density (OD) of 1 represented 40 µg/mL RNA. Purity was assessed using the A260/280 ratios which were generally between 1.8 to 2.1.

2.6 Reverse Transcription Polymerase Chain Reaction (RT-PCR) Prior to reverse transcription, 0.25 – 2 µg of total RNA was diluted to 10 µL with nuclease-free water and treated with 1 unit of DNase1 (Invitrogen) for 30 min at room temperature in 1x DNAse1 buffer (Invitrogen). To stop the reaction, the samples were incubated with 1x final concentration of Stop Solution (Invitrogen) for 10 min at 65 ºC. The RNA was then incubated with 200 ng of random hexamers (Invitrogen) for 5 min at 65 ºC and then cooled on ice. First strand synthesis was performed with 2 U SuperScript III reverse transcriptase, 0.5 mM dNTPs, 10 mM dithiolthrietol (DTT), and 1x first strand buffer (all Invitrogen) in a total reaction volume of 20 µL for 1 hr at 50 ºC. PCR was carried out in 25 µL reactions containing 1x final concentration of PCR buffer (20 mM Tris pH 8.4, 50 mM KCl), 1.5 mM MgCl2, 0.2 mM dNTPs (all Invitrogen), 0.2 µM of each primer (Proligo, Lismore, NSW, Australia), 1 U Platinum Taq DNA Polymerase (Invitrogen), and 1 µL of cDNA template. All primer sequences are listed in Appendix C. Samples were amplified on a PTC-200 Peltier thermal cycler DNA Engine (Bresatec, Adelaide, SA, Australia) using the following parameters: 94 ºC for 4 min, then 30-40

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cycles of 94 ºC for 30 sec, 60 ºC for 30 sec, and 72 ºC for 1 min per kb of amplicon, followed by a final extension step of 72 ºC for 8 min. Samples were mixed with loading buffer (final concentration of 0.025% bromophenol blue, 0.025% xylene cyanol, 3% glycerol) and electrophoresed on 0.75-2% agarose gels prepared with Tris-acetate-EDTA (TAE) buffer (0.09 M Tris pH8.0, 0.001% acetic acid, 2.5 mM EDTA) and containing 0.5 µg/mL ethidium bromide. Approximately 0.5 µg of DNA Marker IX (Roche, Castle Hill, NSW, Australia) was used to compare DNA sizes. Images were captured under UV illumination using a Syngene UV System (Geneworks, Adelaide, SA, Australia).

2.7 Quantitative RT-PCR (QRT-PCR) QRT-PCR was conducted in 96-well plates (Axygen, Quantum Scientific, Murrarie, QLD, Australia) using an ABI PRISM 7000 or 7300 Real-time PCR System (Applied Biosystems, Scoresby, VIC, Australia). Each reaction contained 1x final concentration of SYBR Green PCR Master Mix (Applied Biosystems), 50 nM forward and reverse primer, 2.5 µL of diluted cDNA (1:5 in nuclease-free water) and nuclease-free water to a total volume of 20 µL. The cycling parameters were 95 ºC for 10 min then 40 cycles of 95 ºC for 15 sec, and 60 ºC for 1 min, followed by a dissociation step. Baseline and threshold fluorescence values were manually assigned using ABI PRISM 7000 SDS Software (Applied Biosystems). Gene expression was calculated in three different ways depending on the target gene and experiment. Relative expression compared to a control sample was determined using the comparative CT (ΔΔCT) method. For this approach, the efficiency of primers for the target and housekeeping genes was confirmed using validation experiments according to ABI User Bulletin #2 (Applied Biosystems). In cases where the efficiency of primer sets differed or was not determined, relative gene expression was calculated using standard curves of purified PCR products for the target and reference gene. The standard curves were assigned arbitrary values and relative gene expression was once again normalised to a control sample. To calculate absolute numbers of transcripts, samples were compared to standard curves of PCR products cloned into pGEM-T Easy (Promega, Alexandria, NSW, Australia) which had known copy number. In this instance data was expressed as copies per microgram of RNA.

2.8 Ligation and Cloning of PCR Products PCR products were excised from agarose gels and purified using the Wizard SV Gel and PCR Clean-Up System (Promega). The DNA was then ligated overnight at 4 ºC into pGEM-T Easy (Promega) or pcDNA3.1 Flag-His (Invitrogen) in 1x ligation buffer (40 mM Tris,

10 mM MgCl2, 10 mM DTT, 0.5 mM ATP, pH 7.8) with 1 U T4 DNA ligase (Fermentas, Quantum Scientific, Murrarie, QLD, Australia) and nuclease-free water to 10 µL. Approximate

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molar ratios of 3:1 to 6:1 of DNA insert to vector were used. Once constructs were transformed and purified, the correct sequence of inserts was confirmed using ABI BigDye Terminator sequencing at the Australian Genome Research Facility, University of Queensland, Australia.

2.9 Transformation and Purification of Constructs Constructs were transformed into XL-10 Gold high efficiency competent E. Coli cells (Stratagene, Integrated Sciences, Chatswood, NSW, Australia). Approximately 50 ng of plasmid DNA was incubated with 50 µL of cells on ice for 20 min, heat-shocked at 42 ºC for 90 sec, and then resuscitated in 1 mL of Luria Bertani (LB) broth (1% tryptone, 1% NaCl, 0.5% yeast extract, pH 7.5) for 1 hr at 37 ºC. The solution was then spread onto LB agar plates (LB broth with 1.5% agar) containing 0.1 mg/mL ampicillin for antibiotic selection and colonies were allowed to grow overnight at 37 ºC. For pGEM-T Easy cultures, 1 mM isopropyl-b-D- thiogalactopyranoside (IPTG) and 0.08 mg/mL 5-Bromo-4-chloro-3-indolyl b-D-galactoside (X- GAL) were added for blue/white colour selection.

To purify plasmid DNA, colonies picked from agar plates or samples of frozen glycerol stocks were cultured overnight in LB broth in a shaker at 37 ºC. For maxipreps, large cultures were established from starter cultures incubated for 8 hrs at 37 ºC. Cultures were centrifuged at 6000 x g to pellet cells and plasmid DNA was extracted using Qiagen Miniprep or Maxiprep Extraction Kits (Qiagen, Doncaster, VIC, Australia). DNA concentration and purity was assessed using a

NanoDrop 1000 spectrophotometer with an A260 OD of 1 representing 50 µg/mL DNA and an optimal A260/280 ratio of 1.8.

2.10 Protein Extraction Cells were lysed in buffer containing 10 mM Tris pH 8.0, 150 mM NaCl, 5 mM EDTA, 1% Triton X-100 and 1x complete protease inhibitor cocktail (Roche). The lysates were drawn through a 26-gauge needle several times and then centrifuged at 14 000 x g for 15 min at 4 ºC to remove insoluble material. Protein concentrations were determined using a Bicinchoninic Assay Kit (Pierce, Progen, Darra, QLD, Australia) with bovine serum albumin standards (BSA; 0.1-1 mg/mL). Samples were diluted in PBS to 25 µL, mixed with 200 µL of working reagent, and incubated at 37 ºC for 30 min. Absorbances were measured at 562 nM on a Microplate Reader (Bio-Rad, Gladesville, NSW, Australia).

2.11 Western Blotting Protein samples were diluted in loading buffer (250 mmol/L Tris-HCl, pH 6.8, 2% SDS, 10% glycerol, 20 mmol/L β-mercaptoethanol, 0.01% bromophenol blue) and heated for 5 min at 95

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ºC. Samples and pre-stained molecular weight markers (Bio-Rad) were separated with sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) using 7-14% resolving gels with a 4% stacking layer. Electrophoresis was performed at 80 to 150V in Protean II minigel tanks (Bio-Rad) using the Laemmli buffer system (0.0255 M Tris pH 8.3, 0.25 M glycine, 0.1% SDS). Proteins were then transferred to nitrocellulose membranes (Protran, Schleicher and Schell, Medos, Brisbane, Australia) using a Bio-Rad Transblot apparatus with buffer containing 0.0255 M Tris pH 8.3, 0.25 M glycine and 20% methanol. Membranes were blocked with Odyssey buffer (LI-COR Biosciences, Millennium Science, Surrey Hills, VIC, Australia) and then incubated with primary antibodies diluted in Odyssey buffer for 1 Hr at room temperature or overnight at 4 ºC. All primary antibodies are listed in subsequent chapters. The blots were then washed with tris-buffered saline (0.05 M Tris pH 7.4, 0.25M NaCl) with 0.1% Tween-20 (TBS-T) and incubated with donkey-anti-mouse-800 (Rockland Immunochemicals, Gilbertsville, PA, USA) or goat-anti-rabbit-680 (Invitrogen) fluorescent secondary antibodies diluted 1:10 000 in Odyssey buffer for 1 Hr at room temperature. The membranes were washed with TBS-T and imaged using a LI-COR Odyssey scanner. Odyssey software (LI-COR Biosciences) was used for densitometry and adjusting brightness, contrast and intensity settings.

2.12 Transfections For plasmid DNA transfections, cells were grown to 90% confluence and cultured overnight in antibiotic free media. Plasmid DNA was diluted in 50 µL OptiMem (Invitrogen) per cm2 surface area of the tissue culture plate or flask and incubated for 5 min at room temperature. Lipofectamine 2000 (Invitrogen) was separately incubated with the same volume of OptiMem for 5 min at room temperature. A 1:3 ratio of µg of plasmid DNA to µL of Lipofectamine 2000 was generally used. The amount of plasmid DNA and Lipofectamine 2000 used in each experiment is noted in later chapters. The plasmid DNA and Lipofectamine solutions were subsequently combined and incubated at room temperature for 20 min before the mixture was added to cells. Media was changed after 4-6 Hrs or the following day. A similar protocol was used for siRNA transfections except that cells were only grown to 30% confluence and transfected in serum-free media using Oligofectamine (Invitrogen). Oligonucleotide concentrations are listed in relevant chapters. After 6 hrs of transfection, media containing 30% FCS was added to restore the total FCS concentration to 10%.

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3Chapter 3: Expression and Regulation of Kallikrein-Related Serine Peptidases in Prostate Cancer

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3.1 Introduction The levels of kallikrein-related serine peptidases are associated with patient diagnosis and the molecular pathogenesis of prostate cancer, yet their expression and transcriptional regulation are incompletely defined. KLK2, 3, 4, 14 and 15 are all highly expressed in the prostate, while lower levels of other kallikreins are also present (Harvey et al., 2000, Stephan et al., 2007). In the normal prostate, kallikreins are co-expressed in luminal epithelial cells and secreted into seminal plasma. However, the glandular architecture degenerates during prostate cancer progression. This causes uncontrolled secretion of kallikreins which then leach into the bloodstream leading to higher levels in patient serum. When aberrantly secreted into the tumour microenvironment, kallikreins are also exposed to a new range of substrates compared with seminal plasma, giving them novel pathophysiological functions that may contribute to cancer progression.

With respect to their roles as adjunct biomarkers and functional modulators of the microenvironment, kallikreins should be investigated in combination rather than isolation. KLK3 is not a cancer-specific biomarker, but rather a prostate-specific marker altered by anatomical changes during cancer progression (Lilja et al., 2008). Targeting other prostatic kallikreins is likely to enhance, not replace, the KLK3 test by providing more thorough information and a level of redundancy. Kallikreins have been studied as multiparametric biomarkers for breast, lung, testicular and ovarian cancer and skin disorders (Luo et al., 2003c, Yousef et al., 2003d, Yousef et al., 2004c, Komatsu et al., 2005, Komatsu et al., 2007a, Komatsu et al., 2007b, Planque et al., 2008). Similar studies have not been undertaken for prostate cancer, although KLK2 serum testing has been analysed comprehensively (Lilja et al., 2008, Vickers et al., 2008) and the tissue mRNA levels of KLK11, KLK14 and KLK15 have been separately compared with KLK3 levels in patient serum (Stephan et al., 2003, Stavropoulou et al., 2005, Scorilas and Gregorakis 2006, Lilja et al., 2008, Rabien et al., 2008, Vickers et al., 2008). The proteolytic functions of the kallikreins in cancer progression are similarly interconnected. KLK2, 4-7 and 12-15 have all been shown to activate proKLK3 in vitro or in seminal plasma (Lovgren et al., 1997, Takayama et al., 1997, Vaisanen et al., 1999, Takayama et al., 2001a, Takayama et al., 2001b, Matsumura et al., 2005, Michael et al., 2006, Yoon et al., 2007, Emami and Diamandis 2008). Adding further complexity, many other kallikreins are also activated by other family members, for example KLK4, 5, 12 and 14 all hydrolyse proKLK2 (Michael et al., 2006, Yoon et al., 2007). Furthermore, in vitro studies indicate that many substrates, such as IGFBPs and ECM proteins, are degraded by several kallikreins. The use of kallikreins as multiparametric biomarkers, their activation cascades and redundancy of substrates all suggest that kallikreins should be studied in groups rather than as separate factors.

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The first step in exploring the collective role of kallikreins in cancer progression is to compare their expression in patient tissues and transcriptional regulation in prostate cancer cells. KLK3 levels in prostate cancer have been thoroughly examined. Despite reduced expression at the cellular level, aberrant secretion of KLK3 from malignant glands results in higher levels in the tumour microenvironment and patient serum. In contrast, increased expression of KLK2, KLK4, 14 and 15 in cancer versus benign tissue has been reported (Darson et al., 1997, Darson et al., 1999, Herrala et al., 2001, Yousef et al., 2001c, Obiezu et al., 2002, Stephan et al., 2003, Yousef et al., 2003e, Dong et al., 2005a, Veveris-Lowe et al., 2005). Higher KLK4 expression in prostate cancer is supported by immunohistochemistry and in situ hybridisation experiments but not ELISAs of prostate tissue extracts (Obiezu et al., 2002, Dong et al., 2005a, Obiezu et al., 2005, Klokk et al., 2007, Ramsay et al., 2008a). Increased KLK14 levels were confirmed with immunohistochemistry but not QRT-PCR of laser-capture microdissected samples (Borgono et al., 2003, Borgono et al., 2007b, Rabien et al., 2008). Steroid hormones have been the focus of studies on the transcriptional regulation of kallikreins. Of particular relevance to the prostate, androgen response elements have been identified in the KLK2 and KLK3 promoters and separate studies have indicated that KLK4, 14 and 15 are also up-regulated by androgens in breast or prostate cancer cells (Nelson et al., 1999b, Clements et al., 2001, Korkmaz et al., 2001, Yousef et al., 2001c, Yousef et al., 2002b, Borgono et al., 2003, Yousef et al., 2003e, Xi et al., 2004b, Dong et al., 2005a, Paliouras and Diamandis 2008). However, the notion that prostatic kallikreins are similarly transcriptionally regulated conflicts with reports that their expression profiles in prostate cancer differ; KLK3 is down-regulated while KLK2, 4, 14 and 15 are up- regulated in tumours. One explanation for this paradox is the difficulty in comparing kallikreins from separate studies using different sample types and methodologies. Intra-study discrepancies in the levels of individual kallikreins measured with different techniques have also been noted (Obiezu et al., 2002, Rabien et al., 2008).

A more integrated understanding of kallikreins in prostate cancer is required. In this study, the expression and regulation of KLK2, 3, 4, 14 and 15, the most abundant kallikreins in the prostate, were compared. The expression profiles of these kallikreins were evaluated using QRT- PCR and Western blots of prostate cell lines as well as immunohistochemistry of prostate tissue specimens. These data indicated that KLK2, 3, 4, 14 and 15 are co-expressed. Therefore, the transcriptional regulation of these kallikrein was further scrutinised with particular emphasis on androgens. Finally, other kallikreins which are more lowly expressed were investigated to gain further insight into the regulation of the entire kallikrein locus in prostate cancer.

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3.2 Materials and Methods

3.2.1 Immunohistochemistry Blocks of formalin-fixed paraffin-embedded prostate tissue from radical prostatectomies were provided by Dr Hemamali Samaratunga (Department of Anatomical Pathology, Sullivan Nicolaides Pathology, Brisbane) with QUT institutional ethics approval and sectioned by Dr Ying Dong (QUT). Tissue microrrays (TMAs) with 18 cores per slide (7 BPH, 8 Gleason grade 3-4 cancer, 2 stroma and 1 urothelial cancer) were obtained from the Australian Prostate Cancer Collaboration BioResource. Specimens were dewaxed in xylene (2 x 2 min), rehydrated with consecutive washes of 100%, 90%, 70% and 50% ethanol (2 x 2 min each), rinsed in reverse osmosis (RO) water, and washed in TBS-T (3 x 5 min). Antigen retrieval was performed by microwaving samples in 5% (w/v) urea in 0.1 M Tris pH 9.5 (2 x 5 min). Samples were then washed in TBS-T (3 x 5 min) and incubated in 3% hydrogen peroxide (v/v) and 20% methanol (v/v) in TBS-T for 10 min to quench endogenous peroxidase activity. After further washing (TBS-T, 3 x 5 min), specimens were blocked in 5% non-fat skim milk (w/v) in TBS-T and incubated overnight at 4 ºC with primary antibodies diluted in TBS-T (listed in Appendix D). Primary antibodies were omitted or replaced with normal mouse or rabbit IgG (Dako, Campellfield, VIC, Australia) for negative controls. Slides were washed (TBS-T, 3 x 5 min), incubated with secondary antibodies for 30 min at room temperature (Dako Envision), and rewashed (TBS-T, 3 x 5 min) before 3,3’-diaminobenzidine (DAB), diluted according to the manufacturer’s instructions, was applied (Dako). Brown staining generally developed within 30 sec to 5 min. Samples were washed in running RO water for 5 min, incubated in Mayer’s haemotoxylin (Australian Chemical Reagents, QLD, Australia) for 5 min to counterstain nuclei, and rinsed in RO water for 5 min. Finally, samples were dehydrated in 50%, 70%, 90% and 100% ethanol (2 x 2 min each), cleared in xylene (2 x 2 min) and mounted with DePex mounting medium (Gurr, BPH, Poole, England). Images were captured using an Olympus BX41 light microscope equipped with a Micropublisher 3-3 RTV digital camera controlled with QCapturePro software (Olympus Australia, Brisbane, QLD, Australia).

3.2.2 Analysis of Publicly Available Microarray Data Microarray data previously published by Zhao and coworkers (2005) were downloaded from the Genome Expression Omnibus (www.ncbi.nlm.nih.gov/geo/) using R (www.r-project.org) with

BioConductor (www.bioconductor.org) and GEOquery installed. The Log2 expression values for kallikreins were extracted from the datasets, normalised and median centred using Cluster (Eisen et al., 1998), and displayed as a heatmap using TreeView (http://rana.lbl.gov/EisenSoftware.htm). Microarray data reported by Jia and colleagues (2008)

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were downloaded, analysed and graphed using Microsoft Excel. Average Log2 values for C42B cells treated for 16 hrs with DHT were normalised to cells treated with ethanol vehicle control.

3.2.3 RT-PCR and QRT-PCR Total RNA was extracted from cells and reversed transcribed as previously described (Chapters 2.5 & 2.6). RT-PCR and QRT-PCR were performed according to standard procedures (Chapters 2.6 & 2.7) .All primers are listed in Appendix C.

3.2.4 Clustering Kallikrein mRNA Expression in Prostate Cell Lines Copy numbers of KLK2, 3, 4, 6, 11, 14 and 15 per µg RNA were measured using QRT-PCR with plasmid standard curves of known copy number (Chapter 2.7). Primers are listed in Appendix C .The lower detection limit was set at 1 x 102 copies per µg RNA. Average linkage clustering of Log10 kallikrein copy numbers was performed for genes and cell lines with Cluster and results were visualised with TreeView. More intense shades of red represent higher kallikrein expression.

3.2.5 Western Blotting Whole cell lysates were prepared and analysed with Western blots using previously described methods (Chapters 2.10 & 2.11). Relative protein levels were qualitatively compared using GAPDH as a loading control. All primary antibodies are listed in Appendix D.

3.2.6 Steroid Hormone Treatments LNCaP and 22Rv1 cells were seeded onto 6-well plates in RPMI 1640 containing 10% FCS and allowed to attach for 24 to 48 hrs or until approximately 70% confluent. The culture media was then changed to RPMI 1640 containing 10% charcoal-stripped FCS (CSS), which is devoid of steroid hormones, for 72 hrs. CSS was always added to phenol red-free RPMI 1640 medium. The synthetic androgen, R1881 (methyltrienolone, Sigma), was prepared in 100% AR-grade ethanol for a 1 mmol/L stock solution. Cells were treated with 1 nM R1881 or ethanol vehicle control diluted in 10% CSS containing media for 24 hrs unless otherwise indicated. The final alcohol concentration was 0.1% or less. Bicalutamide (Astra Zeneca, Brisbane, Australia) was added 2 hrs prior to R1881 for AR antagonist experiments. Any changes to these parameters are noted in figure legends accompanying each experiment. RNA for QRT-PCR and protein lysates for Western blot analysis were extracted from treated cells as outlined in Chapter 2.10. RNA for the R1881 time course and bicalutamide treatments was provided by Dr John Lai, QUT. Ms Melanie Hunt, QUT, performed the cell culture steps of the bicalutamide experiments.

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3.2.7 Steroid Hormone Deprivation LNCaP and 22Rv1 cells were seeded into 6-well plates in RPMI 1640 containing 10% FCS and grown to 30% confluency. Cells were then cultured in RPMI 1640 with 10% CSS for 1 to 10 days with media changes every 2 to 3 days. Cellular morphology was imaged with a Leitz Laborlux S light microscope fitted with a Nikon DXM 1200C digital camera using ACT-1C software (Leica, Gladesville, NSW, Australia).

3.2.8 Collection and Concentration of Conditioned Media T80 culture flasks were seeded with 2 x 106 LNCaP and DU145 cells in RPMI 1640 with 10% FCS. After 24 hrs, media was changed to RPMI 1640 containing 10% CSS for 72 hrs. LNCaP cells were then treated with 1 nM R1881 or ethanol control in 2% CSS for 24 hrs. DU145 cells were also cultured in 2% CSS with vehicle control. This media was removed and cells were cultured for a further 24 hrs in fresh 2% CSS containing media with ethanol or R1881. The conditioned media were collected, centrifuged at 1500 x g for 5 min to remove cellular debris, spiked with protease inhibitor cocktail to 1x final concentration, and stored on ice. To account for differences in proliferation, cells were counted and conditioned media volumes adjusted with 2% CSS media so that the ratio of cells per millilitre, typically 5 x 105 cells/mL, was equal among treatments. Conditioned media were then concentrated 75 to 80-fold using 10 kDa cut-off filter microfuge columns with successive 14,000 x g spins for 20 min at 4 ºC. Final samples were stored at -80 ºC.

3.2.9 Androgen Receptor Knock Down LNCaP cells were seeded at a density of 1 x 105 cells per well in 6-well plates, grown for 48 hrs so they reached approximately 30% confluence, and cultured in antibiotic-free RPMI 1640 with 10% CSS for 72 hrs. Immediately prior to transfection, media was replaced with 800 µL serum- free RPMI 1640. Cells were transfected with a previously described siRNA duplex targeting the AR coding region (sense, 5’ ACG UUU ACU UAU CUU AUG CdTdT 3’; antisense, 5’ GCA UAA GAU AAG UAA ACG UdTdT 3’) or with mock (no siRNA) and non-targeting controls (sense, 5’ AAU UUU ACU CGC UCG AUU UdTdT 3’; antisense, 5’ AAA UCG AGC GAG UAA AAU UdTdT 3’) (Sigma-Proligo, Sydney, Australia) (Jia et al., 2006) using the standard protocol (Chapter 2.13). For each well, 100 pmol of siRNA (5 µL of 20 pmol/L solution) was mixed with 180 µL OptiMEM (Invitrogen), and 4 µL of Oligofectamine (Invitrogen) was added to 11 µL OptiMEM. The final concentration of siRNA was 100 nM once 200 µL siRNA:oligofectamine mix was added to the cells. After 4 hrs, 500 µL of RPMI 1640 containing 30% CSS was added for 10% final serum concentration. Approximately 48 hrs after transfection, cells were treated with 1 nM R1881 or ethanol control for 48 hrs.

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3.2.10 RNA Stability Assay LNCaP cells (2 x 105/well) were seeded onto 6-well plates and allowed to attach for 24 hrs in RPMI 1640 medium containing 10% FCS. Cells were then cultured in 10% CSS containing media for 72 hrs and treated with 1 nM R1881 or ethanol control for 24 hrs. Transcription was halted with 10 µg/mL Actinomycin D (Sigma), a potent inhibitor of the transcription initiation complex (Sobell 1985). Cells were collected for RNA extraction after 2, 4, 6 and 8 hrs of treatment. The dose and duration of Actinomycin D treatments are consistent with a previous study (Lee et al., 2007). Cell death occurred at longer timepoints such as 20 hrs. The amount of mRNA remaining at each time point was measured using QRT-PCR with standard curves of known copy number (Chapter 2.7). Values for R1881 and ethanol treated cells were compared separately. Transcript copy numbers at time 0 samples were set at 100%.

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3.3 Results

3.3.1 Kallikrein 2, 3, 4, 14 and 15 are Co-expressed in a Subset of Prostate Cell Lines Seven prostate cell lines with a spectrum of aggressiveness and androgen responsiveness were used to compare KLK2, 3, 4, 14 and 15 mRNA and protein expression. The AR status of each cell line was confirmed with RT-PCR (Figure 3.1A). Copy numbers of KLK2, 3, 4, 14 and 15 transcripts in each cell line were measured using QRT-PCR. For further comparisons, KLK6 and KLK11 levels were also quantified. KLK6 is differentially expressed in several malignancies including skin, colon and ovarian cancer (Yousef et al., 2003d, Nagahara et al., 2005, Ogawa et al., 2005, Klucky et al., 2007), while KLK11 expression is reportedly increased in prostate cancer (Nakamura et al., 2003c, Stavropoulou et al., 2005, Scorilas and Gregorakis 2006). Expression levels varied widely between kallikreins and cell lines, spanning seven orders of magnitude from below the detection limit to 1.56 x 108 copies of KLK3 per microgram of total RNA in MDA-PCa-2b cells (Figure 3.1A). This data was clustered to investigate patterns in the expression levels of these kallikreins. No relationship between kallikrein expression and the aggressiveness of the prostate cell line was apparent. However, KLK2, 3, 4 and 15 grouped together and their expression significantly correlated with positive AR status. KLK2 and KLK3 were not expressed in cell lines that lack AR, whereas low levels of KLK4 in PC3 cells and KLK15 in DU145 cells were detected. Notably, KLK14 was the only gene expressed in all seven cell lines, albeit at more modest levels. Although there was no clear pattern for KLK11, KLK6 expression appeared to negatively correlate with AR status, although this observation was confounded by the lack of KLK6 expression in androgen insensitive DU145 cells. Analysis of a previously reported microarray dataset of five androgen responsive cell lines (LAPC-4, MDA- PCa-2a, MDA-PCa-2b, 22Rv1 and LNCaP) and two androgen insensitive cell lines (PC3 and DU145) yielded similar expression profiles for KLK2, 3, 4, 6 and 11 (Zhao et al., 2005a) (Figure 3.1B). The microarray chips used in this study did not have KLK14 or KLK15 probes. These results show that KLK2, 3, 4, 14 and 15 are co-expressed in a subset of prostate cell lines, although KLK14 has a broader expression profile.

The expression profiles of KLK2, 3, 4, 14 and 15 were also examined at the protein level using Western blots of whole cell lysates from the seven prostate cell lines. Specific immunoreactive bands of appropriate size, 30-32 kDA for KLK2, KLK3 and KLK15 and 25kDA for KLK14, were detected (Figure 3.2). Multiple KLK2 and KLK3 bands represent differently glycosylated isoforms as previously reported (Wang et al., 1999a, Wang et al., 1999b). Non-specific higher molecular weight bands were observed in KLK14 and KLK15 blots. Protein expression profiles

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Figure 3.1. Comparison of Kallikrein Expression Levels in Prostate Cell Lines A - KLK2, KLK3, KLK4, KLK6, KLK11, KLK14 and KLK15 expression in 22Rv1, LNCaP, MDA-PCa-2b, DU145, PC-3, RWPE-2 and RWPE-1 prostate cell lines as determined with QRT-PCR. The heatmap was generated using log copy numbers of transcripts per µg RNA using Cluster and Treeview. More intense shades of red indicate higher expression. AR expression in 22Rv1, LNCaP and MDA-PCa-2b cells was confirmed with RT-PCR. KLK2, KLK3, 4 and 15 expression has a significant positive correlation with AR status (* P<0.05, n=3, Spearman’s Rank Correlation). B – A heatmap of kallikrein expression in prostate cell lines based on microarray data reported by Zhao and co-workers (2005). Red indicates higher expression compared with pooled contol cDNA whereas green indicates lower expression. Grey boxes indicate missing values that were filtered out of analyses due to very low expression. Of the KLK3 probes, 4 had lower values, possibly due to poor hybridisation.

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Figure 3.2. Expression of Kallikrein Proteins in Prostate Cell Lines Western blots were used to compare the expression of kallikreins in whole cell lysates from RWPE-1, RWPE-2, LNCaP, 22Rv1, PC-3, DU145 and MDA-PCa-2b prostate cell lines. Arrows indicate bands specific for each kallikrein. Between 10 µg (KLK2/KLK3) and 40 µg (KLK14/KLK15/AR) of lysate was used per sample. Representative data from 2 experiments is shown. GAPDH was used as a loading control for all blots. The GAPDH panel in this figure is from the AR blot.

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for each kallikrein matched the QRT-PCR results. KLK2, KLK3 and KLK15 were most highly expressed in androgen responsive LNCaP, 22Rv1 and MDA-PCa-2b cells. AR protein expression was confirmed in these cell lines including the low molecular weight AR variants in 22Rv1 cells (Dehm et al., 2008). Some non-specific bands with slightly lower molecular weight than AR were also observed. KLK15 Western blots confirmed the low level of KLK15 expression in DU145 cells measured with QRT-PCR. KLK14 was detected in all cell lines except RWPE-1 and RWPE-2 cells which had the lowest levels of KLK14 mRNA. Although eight different primary antibodies were trialled, no specific KLK4 immunoreactivity was observed (data not shown). Any bands that appeared for cell lines that do not express KLK4 mRNA were deemed to be non-specific.

3.3.2 Kallikrein 2, 3, 4, 14 and 15 are Co-expressed in Benign and Malignant Glandular Epithelial Cells in Prostate Tissue Considering that KLK2, 3, 4, 14 and 15 are co-expressed in prostate cell lines, immunohistochemistry was used to examine their expression and localisation in prostate tissue specimens. At least two different primary antibodies targeting each kallikrein were used. The well established staining pattern of KLK3 was observed with three different primary antibodies, KLK3A, B and C, which exhibited immunoreactivity restricted to the cytoplasm of prostate epithelial cells (Figure 3.3A-C). KLK3 was particularly concentrated at the apical surface of luminal epithelial cells, as expected for a secreted protein. KLK15 was localised to the cytoplasm of epithelial cells with two different KLK15 primary antibodies (KLK15A and KLK15B) (Figure 3.3D-F). Additionally, nuclear staining of epithelial cells was frequently observed with both KLK15 antibodies, although the abundance of nuclear staining varied within and between samples. For example, the glands shown in Figure 3.3E and Figure 3.3F were 450 µm apart in the same section probed with the KLK15B antibody. There was no apparent correlation between KLK15 localisation and the pathology of glands. Alternative splicing gives rise to several variant kallikreins with altered cellular localisation from the traditional transcripts, including nuclear localisation (Tan et al., 2006). Interestingly, several KLK15 mRNA variants were detected with RT-PCR in range of prostate cell lines (Figure 3.3G). These variants have all been observed in other cell types; however the cellular localisation of these isoforms, if translated, is unknown (personal communication Dr Olivia Tan, QUT).

Cytoplasmic, apically concentrated localisation of KLK2 was observed in epithelial cells probed with the KLK2A primary antibody (Figure 3.4A). There was no staining of endothelial cells or stroma. KLK14 expression was detected in muscle cells (Figure 3.4B and inset) as well as luminal epithelial cells (Figure 3.4C & D) with the KLK14A and KLK14B primary antibodies. This is consistent with the high levels of KLK14 in the prostate and skeletal muscle

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Figure 3.3. KLK3 and KLK15 Expression in Prostate Tissue A-C – Immunohistochemistry of KLK3 using three different anti-KLK3 primary antibodies. D-F – KLK15 staining with two different antibodies. E & F are from the same patient section, approximately 450 µM apart. G – RT-PCR of KLK15 in prostate cancer cell lines showing the expression of several splice variants. The main band at 686 bp (arrow) is full length KLK15 (personal communication Dr Olivia Tan, QUT). The 669 bp band that is only partially separated from full length KLK15 is a variant with intron 3 retention and exon 4 deletion. The other amplified variants have partial exon 3 deletion (586 bp), complete exon 4 deletion (549 bp), and a combination of partial exon 3 and complete exon 4 deletion (431 bp). The extra bands at approximately 550 and 270 do not correspond to any previously described KLK15 variants.

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Figure 3.4. KLK2 and KLK14 Expression in Prostate Tissue A – An example of KLK2 immunohistochemistry with staining of glandular epithelial cells. B – Staining of smooth muscle cells in a prostate specimen with the KLK14B antibody. C & D – KLK14 immunoreactivity in prostate epithelial cells with KLK14A and KLK14B antibodies. E – KLK2 expression in a specimen with adjacent regions of prostatic intraepithelial neoplasia (PIN) and Grade 3 cancer (Ca). There is weaker staining in malignant glands. F –The same sample probed with the KLK14B primary antibody. Staining intensity is similar among glands.

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observed with Northern blots of tissue RNA extracts (Hooper et al., 2001). Adjacent cancer and benign glands were present in serial sections stained with the KLK2A and KLK14B antibodies (Figure 3.4E&F). In this instance, there were similar levels of KLK2 and KLK14 between glands.

Two main protein variants of KLK4, with different cellular localisation, have been reported in prostate cancer (Dong et al., 2005a). KLK4-254 is cytoplasmically localised and has the typical kallikrein zymogen structure, a predomain that targets the protein for secretion and the extracellularly cleaved prodomain for enzyme activation. In comparison, KLK4-205 lacks the pre- and pro-regions and is nuclear localised. In this study, three different sets of KLK4 primary antibodies, KLK4A, B & C, detected KLK4 in the cytoplasm of luminal epithelial cells with the most intense staining towards the apical surface (Figure 3.5A, B & C). KLK4 is also expressed in basal cells (Figure 3.5D), as noted in previous studies using in situ hybridisation (Xi et al., 2004b). Previously reported nuclear KLK4 immunoreactivity was not detected (Dong et al., 2005a, Klokk et al., 2007). Several other studies also failed to observe nuclear KLK4 in prostate and other hormone-dependent tumour samples (Day et al., 2002, Obiezu et al., 2002, Davidson et al., 2005, Davidson et al., 2007). In serial sections probed with KLK4B and KLK4C primary antibodies, greater KLK4 expression was observed in benign and PIN glands than adjacent regions of Grade 4 cancer (Figure 3.5E & F). In contrast, other studies using immunohistochemistry have reported increased KLK4 expression in prostate cancer compared with benign glands (Dong et al., 2005a, Veveris-Lowe et al., 2005, Klokk et al., 2007, Ramsay et al., 2008a). In two other reports, no difference in the intensity of KLK4 immunoreactivity between cancer and benign glands was noted (Day et al., 2002, Obiezu et al., 2002). Future studies using larger cohorts and multiple KLK4 antibodies may clarify these discrepancies.

Other primary antibodies targeting each kallikrein were also tested but were unsuitable for immunohistochemistry. High background staining of stroma was observed with KLK2B and KLK14E antibodies while KLK14D cross-reacted with recombinant KLK3 and KLK4 in Western blots (data not shown). No staining was observed with KLK3D, KLK4D, KLK14C and KLK15C antibodies even at 1:50 dilutions, or when antigen retrieval was omitted or performed under different conditions. These antibodies were designed to block kallikrein enzyme activity and presumably bind to conformational epitopes that are destroyed by tissue fixation.

To compare the expression profiles of prostatic kallikreins in BPH and cancer, serial sections of a small tissue microarray were probed with KLK2, 3, 4, 14 and 15 primary antibodies. Multiple primary antibodies targeting KLK4, KLK14 and KLK15 were used because the expression patterns of these kallikreins are less well characterised than KLK2 and KLK3. KLK2A, KLK3C, KLK4B, KLK4C, KLK14A, KLK14B, KLK15A and KLK15B primary antibodies were chosen

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Figure 3.5. KLK4 Expression in Prostate Tissue A-C – Immunohistochemical staining of prostate epithelial cells with three different KLK4 antibodies, KLK4A, KLK4B and KLK4C. D – A magnified version of C with basal cells staining for KLK4 indicated with arrows. E & F – Serial sections of a sample probed with KLK4B and KLK4C primary antibodies with more intense staining in areas of prostatic intraepithelial neoplasia (PIN) compared with adjacent Gleason Grade 4 cancer (Ca).

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because they produce minimal background staining of stroma and do not cross-react with peptides spanning the same domains of other kallikreins (TriplePoint Biologics product specifications; www.triplepointbiologics.com). KLK4A is a combination of two antibodies raised against the N-terminus and mid-region of KLK4. There are published reports of imunohistochemistry with prostate specimens for these antibodies and their specificity has been examined with peptide pre-absorption experiments (Harvey et al., 2003, Dong et al., 2005a, Veveris-Lowe et al., 2005, Ramsay et al., 2008a).

Results for the nine anti-kallikrein primary antibodies and rabbit isotype antibody control are summarised in Figure 3.6. Two cores that only consisted of stromal cells had negligible immunoreactivity. Slight over-staining of KLK3 and KLK4B slides led to higher background colouration, while KLK4A, KLK14A and KLK15A-probed sections are under-stained. Staining intensity was less than the isotype control for 25% of cores with KLK14A and 50% of samples with KLK4A and KLK15A antibodies. The small number of cores and lack of aggressive Gleason Grade 5 cases precludes the analysis of changes during prostate cancer progression for each kallikrein. There is more variation in staining within the set of cancer samples than between the benign and cancer cores. Nevertheless, the expression of different prostatic kallikreins can be compared across the serial sections. Figure 3.7 focuses on 2 regions each of benign and malignant glands for which cores were present on all 10 slides. Each kallikrein was expressed in both benign and malignant glands. The relative intensity of immunoreactivity between kallikreins was similar in benign and cancer samples with robust KLK2 and KLK3 expression in all cases. KLK4, KLK14 and KLK15 staining was more variable. Among the benign cores, there was strong KLK4 but not KLK14 and KLK15 expression in specimen #1, while low KLK4 but high KLK14 and KLK15 expression was observed in specimen #2. Among the malignant samples, all prostatic kallikreins stained strongly in specimen #3, whereas KLK4, KLK14 and KLK15 were more lowly expressed in specimen #4. These results demonstrate that although there is sample to sample variation, KLK2, 3, 4, 14 and 15 expression is maintained in prostate cancer and these kallikreins are co-expressed by benign and malignant prostate epithelial cells.

3.3.3 Prostatic Kallikreins are Differentially Regulated by Androgens in LNCaP and 22Rv1 Prostate Cancer Cells From the co-expression of KLK2, 3, 4, 14 and 15 in prostate cell lines and tissue specimens it can be proposed that the transcriptional regulation of these kallikreins may be similar. The highest levels of prostatic kallikrein expression were in androgen responsive LNCaP, 22Rv1 and MDa-PCa-2b cells. Furthermore, the androgen regulation of KLK2 and KLK3 is well characterised (Kim and Coetzee 2004). Therefore, the role of androgens in regulating KLK2, 3, 4, 14 and 15 expression was further examined with particular emphasis on comparing changes

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Figure 3.6. Kallikrein Immunohistochemistry with Tissue Microarrays Immunohistochemisty with KLK2, KLK3, KLK4A, KLK4B, KLK4C, KLK14A, KLK14B, KLK15A, KLK15B and rabbit isotype control antibodies on serial sections of prostate tissue. Cores of benign prostate hyperplasia (BPH), cancer and stroma only are grouped together. There was also one core of urothelial cancer. Spaces represent missing cores in particular sections. The scale bar (100 µm) is the same for all images.

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Figure 3.7. Kallikrein Immunohistochemistry on Serial Sections of Prostate Tissue Higher magnification images of KLK2, 3, 4, 14, 15 and isotype control staining in serial sections of tissue microarray cores. Specimens #1 and #2 are benign while specimens #3 and #4 are Gleason grade 4 cancer. Scale bars equal 25 µm.

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between these kallikreins. LNCaP cells, the most commonly used model for the AR signalling axis, were cultured in CSS-containing media, which is devoid of hormones, to reduce the expression of androgen regulated genes to basal levels. The hormone deprived LNCaP cells were then treated for 3, 6 and 24 hrs with 1 nM R1881, a synthetic androgen, which is more stable than physiological ligands such as testosterone and DHT. KLK2, 3, 4 and 15 mRNA expression was significantly increased with R1881 treatment and time when compared to cells treated with ethanol solvent control (Figure 3.8; P<0.001). A trend of increased kallikrein expression after 6 hrs of R1881 treatment, which was significant for KLK4 (P<0.01) and KLK15 (P<0.05), suggests specific responses to androgens rather than to general changes in cellular phenotype. Increased expression of 46-fold for KLK2, 30-fold for KLK3, 3.6-fold for KLK4 and 2.2-fold for KLK15, was observed in LNCaP cells exposed to androgens for 24 hrs. In contrast, a significant 73% decrease in KLK14 expression was measured (P<0.05). This surprising result differs from reports that KLK14 expression is stimulated by androgens in breast cancer cells (Borgono et al., 2003, Yousef et al., 2003a, Paliouras and Diamandis 2008).

To further compare prostatic kallikrein expression, LNCaP cells were treated for 24 hrs with 0.01 to 10 nM R1881. Maximal changes in KLK2, 4, 14 and 15 expression were observed with 1 nM R1881, which is equivalent to physiological androgen concentrations (Figure 3.9). Significant increases in KLK4 (P<0.05) and KLK15 (P<0.01) expression were also measured in LNCaP cells treated with 0.1 nM R1881. The trend of reduced kallikrein expression with 10 nM R1881 is consistent with the biphasic response of LNCaP cells to androgens that has been noted in other studies. Although the magnitude of fold changes differ from the time course experiment, 295 for KLK2, 195 for KLK3, 25 for KLK4, 12 for KLK15 and a 42% decrease in KLK14 expression, the relative changes between kallikreins was consistent. KLK2 and KLK3 were strongly up-regulated, KLK4 and KLK15 levels were moderately increased and KLK14 expression was repressed. Androgens also induced corresponding changes in the protein levels of prostatic kallikreins. DU145 cells where used as a negative control for all Western blots because they do not express KLK2, 3 and 4 and only low levels of KLK14 and KLK15. KLK2, 3 and 15 levels were increased in lysates of LNCaP cells treated with 0.01 to 10 nM R1881 for 24 hrs and maintained in cells treated for three and seven days (Figure 3.10A). Greater levels of KLK2, 3 and 15 were also present in the conditioned media of LNCaP cells treated with R1881 compared with ethanol control (Figure 3.10B). Intriguingly, there was an approximate 10 kDa increase in the size of KLK15 in conditioned media compared with whole cell lystates, possibly due to glycosylation at two predicted sites. No change in KLK14 expression in whole cell lysates was observed after one or three days, although there was a discernable decrease after seven days of R1881 treatment (Figure 3.10A). KLK14 was not detected in conditioned media samples.

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Figure 3.8. Kallikrein mRNA Expression in LNCaP Cells with a Time Course of R1881 Treatment LNCaP cells were cultured in media containing 2% CSS for 48 hrs and then treated with ethanol or 1 nM R1881 for 0, 3, 6 and 24 hrs. QRT-PCR was used to determine kallikrein levels with data normalised to the ethanol control for each time point. Data represents average fold changes of R1881 treatment compared with ethanol for each time point. Error bars represent SEMs (n=2, ### denotes a significant effect of time, R1881 treatment and both factors combined P<0.001, 2 Way ANOVA, asterisks indicate significant differences between R1881 and ethanol for particular time points using Tukey’s posthoc analysis; * P<0.05, ** P<0.01, ***P<0.001).

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Figure 3.9. Dose Dependent Changes in Kallikrein Expression in R1881 Treated LNCaP Cells LNCaP cells were grown in 10% CSS containing media for 72 hrs and then treated with ethanol or 0.01 to 10 nM R1881 for 24 hrs. Changes in kallikrein expression measured with QRT-PCR are displayed as average fold changes versus ethanol with error bars representing SEMs (n=3, One Way ANOVA with Tukey’s posthoc analysis; *P<0.05, **P<0.001, ***P<0.001 compared to 0 dose).

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(Figure 3.10, see over)

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Figure 3.10. Kallikrein Protein Levels in LNCaP Cells Treated with R1881. A - Changes in the protein level of kallikreins in whole cell lysates. LNCaP cells were cultured in charcoal-stripped serum containing media for 72 hrs and then treated with ethanol or 0.01 to 10 nM R1881 for 1 day (left panel), 3 days (middle panel) or 7 days (right panel). 20 µg (KLK2/KLK3) to 40 µg of protein was loaded per lane. GAPDH was used as a loading control (the panels in this figure correspond to the KLK2 blots). Representative data from 2 (7 days) or 3 (1 and 3 days) separate experiments are shown. DU denotes DU145 lysates which were used as a negative control. B - Levels of secreted kallikreins in R1881-treated LNCaP conditioned media. LNCaP and DU145 cells were cultured in RPMI 1640 containing 10% CSS for 72 hrs and then treated with ethanol or 1 nM R1881 in 2% CSS containing media for 24 hrs. This media was replaced with fresh RPMI 1640 with 2% CSS and ethanol or R1881 which was collected after an additional 24 hrs. The treated cells were counted and the volumes of conditioned media adjusted with 2% CSS RPMI to account for differences in proliferation and to ensure equal numbers of cells per mL. The conditioned media was concentrated 40 to 80 fold using 10 kDa cut-off filter spin columns and the equivalent of 400-1500 µL of conditioned media was analysed with Western blots. Representative results from 1 of 3 experiments are shown.

Regardless of androgen treatment, KLK4 expression was not observed in LNCaP whole cell lysates (data not shown). In contrast, KLK4 was successfully detected in the concentrated conditioned media of LNCaP cells treated with 1 nM R1881 but not ethanol and, also, not in DU145 conditioned media (Figure 3.10B). A non-specific higher molecular weight band was present in all cell lines. This must correspond to full length KLK4-254, because N-terminally truncated KLK4-205 variant lacks the signal peptide necessary for secretion.

The nuclear localised KLK4-205 variant has not been detected with immunohistochemistry or Western blots in this study (Figures 3.5, 3.6, 3.7, 3.10B and data not show). In contrast, KLK4- 254, was detected as cytoplasmic staining with immunohistochemistry and in LNCaP conditioned media, but not whole cell lysates, with Western blotting (Figures, 3.5, 3.6, 3.7, 3.10B and data not shown). To further characterise these variants, their expression and androgen regulation in LNCaP cells was compared with QRT-PCR. KLK4-254 and KLK4-205 both contain exons 2 to 5, but exon 1 is only present in KLK4-254. Therefore, KLK4-254 copy numbers were measured using primers targeting exon 1 and 2. KLK4-205 levels were calculated by subtracting KLK4-254 copy numbers from the total number of transcripts with exon 2 to 3. As shown in Figure 3.11, the expression of both transcripts is significantly up-regulated by R1881 treatment with similar fold changes compared with ethanol control (KLK254, P<0.01, KLK205, P<0.05). Notably, KLK4-254 is lowly expressed and 1000-fold less abundant than

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KLK4-205. Nevertheless, KLK4-254 can be detected at the protein level, unlike the more abundant KLK4-205 variant (Figure 3.10B).

Changes in prostatic kallikrein expression were also measured in 22Rv1 cells, a model of androgen-independent, yet androgen-responsive, prostate cancer. Similar to LNCaP cells, expression of KLK2, 3, 4 and 15 was significantly stimulated by R1881 treatment (KLK2; P<0.001, KLK3, 4, 15; P<0.01) although the responses were much smaller in magnitude (Figure 3.12). A 3-fold increase in KLK2 expression was the largest change observed. KLK14 was down-regulated by R1881 treatment to a lesser extent than in LNCaP cells with a 15% decrease in expression. Together with the LNCaP mRNA and protein expression data, these results demonstrate that although KLK2, 3, 4, 14 and 15 are co-expressed, they are differentially regulated by androgens in prostate cancer cells.

3.3.4 Prostatic Kallikreins are Differentially Regulated by Hormone Deprivation Since the growth and survival of prostate cancer cells is initially androgen dependent, surgical and chemical castration are commonly used to manage prostate cancer progression. Changes in prostatic kallikrein expression in response to androgen withdrawal were investigated and compared to the previous experiments where androgens were added to prostate cancer cells. Surgical castration, known as bilateral orchiectomy, reduces circulating androgen levels in men by 90-95%. This was modelled by culturing LNCaP and 22Rv1 cells in media supplemented with CSS, which is devoid of steroid hormones. KLK2, 3, 4 and 15 mRNA levels were significantly reduced in LNCaP cells after just 24 hrs in CSS-containing media (Figure 3.13A, P<0.001). There was a sharper decrease in the expression of KLK2 and KLK3, which were more strongly stimulated by R1881 treatments in previous experiments than KLK4 and KLK15. After 10 days of hormone deprivation, kallikrein expression in LNCaP cells was reduced by 75% for KLK4, 94% for KLK15 and greater than 99% for KLK2 and KLK3. The levels of KLK2, 3 and 15 proteins in LNCaP whole cell lysates also were decreased (Figure 3.13E). The reductions, particularly for KLK15, were less extreme than those observed with QRT-PCR, suggesting that kallikrein protein is more stable than mRNA. In contrast to other prostatic kallikreins, KLK14 mRNA and protein expression was up-regulated by hormone deprivation of LNCaP cells (Figure 3.13B & E). Indeed, there was a significant 3.6-fold increase in KLK14 mRNA expression after 10 days in CSS (P<0.01). More modest changes were once again observed with 22Rv1 cells. KLK2 and KLK3 expression were consistently down-regulated at all time points (Figure 3.13C). However, a significant decrease in KLK4 and KLK15 was not measured in 22Rv1 cells until 4 days of hormone deprivation (P<0.01), unlike 1 day for LNCaP cells (Figure 3.13C). The trend of increased KLK14 expression with 1 to 7 days of hormone deprivation was not statistically significant.

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Figure 3.11. Expression of Kallikrein 4 Variants in LNCaP Cells Treated with R1881. QRT-PCR targeting exons 1-2 or 2-3 of KLK4 was used to calculate copy numbers of transcripts in LNCaP cells treated for 24 hrs with ethanol or 1 nM R1881. Average relative copies numbers compared with 18S are shown with SEM (n=3, t test, *P<0.05, **P<0.01).

Figure 3.12. Changes in Kallikrein Expression in 22Rv1 Cells Treated with R1881. QRT-PCR was used to measure gene expression changes in 22Rv1 cells cultured in media with 10% CSS for 72 hrs and then ethanol or 1 nM R1881 for an additional 24 hrs. Average fold changes in kallikrein expression normalised to ethanol are displayed with SEM (n=5, t test, * P<0.05, ** P<0.01, ***P<0.001).

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(Figure 3.13, see over)

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Figure 3.13. Kallikrein Expression in Hormone-Deprived LNCaP and 22Rv1 Cells. LNCaP and 22Rv1 cells were cultured in media containing 10% CSS for 0, 1, 4, 7 and 10 days. All QRT-PCR results (A-D) are plotted as average fold changes normalised to 0 days with error bars representing SEMs (n=3). A - KLK2, 3, 4 and 15 expression in LNCaP cells (#: KLK2, KLK3, KLK4 and KLK15 P<0.001). B – KLK14 expression in hormone-deprived LNCaP (**P<0.01). B Inset - T47D cells were cultured in media containing 10% FCS or 10% CSS for 4 days and copy numbers of KLK14 compared with 18S were measured using QPCR. Average relative copy numbers are shown with SEM (n=3, t test, p<0.05). C – Kallikrein expression in 22Rv1 cells (a: KLK2 P<0.01, KLK3 P<0.05; b: KLK2 and KLK3 P<0.001, KLK4 and KLK15 P<0.01; c: KLK2 and KLK3 P<0.001, KLK15 P<0.05). D – AR expression in LNCaP and 22Rv1 cells (*P<0.05). E - Representative Western blots from 2 experiments are shown with 20 µg (KLK2, KLK3, β-catenin and NSE) to 40 µg (KLK14, KLK15 and AR) of whole cell lysate per lane. All membranes were reprobed for GAPDH to confirm equivalent loading. GAPDH from the KLK3 blot is shown. F - Phase contrast light microscope images depicting the increasingly neuroendocrine-like morphology of the LNCaP cells cultured in 10% CSS for 0 to 10 days.

KLK14 expression was also measured in T-47D breast cancer cells cultured in CSS for 4 days. Androgens up-regulate KLK14 expression in this cell line in comparison to the down-regulation in LNCaP cells observed in this study (Yousef et al., 2003a, Paliouras and Diamandis 2008). Accordingly, KLK14 expression was significantly down-regulated rather than stimulated by hormone deprivation of T47D cells (Figure 3.13B inset, P<0.05).

In addition to the changes in kallikrein expression, hormone deprivation altered the morphology and phenotype of LNCaP cells. Prostate cancer cells undergo neuroendrocrine-like transdifferentiation as a survival mechanism in response to androgen withdrawal (Yuan et al., 2007). In keeping with this phenomenon, hormone deprived LNCaP cells had irregular morphology with narrower cell bodies and more numerous processes (Figure 3.13F) . Furthermore, the neuroendocrine marker, neuron-specific enolase, was up-regulated, although it was also expressed in basal conditions (Figure 3.13E). β-catenin, which has a functional role in inducing neuroendocrine-like transdifferentiation in the absence of androgens (Yang et al., 2005b), was also increased after 10 days of hormone deprivation (Figure 3.13E). It is possible that, like the kallikreins, changes in neuron-specific enolase and β-catenin mRNA levels would be more pronounced than for the protein. AR mRNA expression was significantly increased in LNCaP cells cultured in CSS-containing media for 10 days (Figure 3.13D, P<0.05). However, the abundance of AR protein was reduced by hormone deprivation because androgens support the post-transcriptional stabilisation of AR (Figure 3.13E). Hormone deprivation did not change

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the AR, neuron-specific enolase or β-catenin expression or cellular morphology of 22Rv1 cells (Figure 3.13D and data not shown) which are less reliant on androgens for growth and survival than LNCaP cells.

AR signalling is also inhibited in prostate cancer patients through chemical castration with compounds such as bicalutamide. This AR antagonist allows the nuclear translocation and binding of AR to the promoters of target genes. However, bicalutamide-bound AR does not stimulate transcription because it fails to recruit transcriptional co-activators (Masiello et al., 2002, Hodgson et al., 2007). To determine the effect of bicalutamide on kallikrein expression, LNCaP cells were treated for 24 hrs with 1 nM R1881 and increasing concentrations of the AR antagonist. In the absence of bicalutamide, R1881 significantly increased KLK2, 3, 4 and 15 expression (Figure 3.14A, P<0.001). The addition of 10 µM bicalutamide significantly reduced KLK2, 3 and 4 expression (KLK2, P<0.01, KLK3 P<0.05, KLK4 P<0.001), whereas KLK15 was not significantly down-regulated until 50 µM bicalutamide was added (P<0.01). KLK2, 3, 4 and 15 expression reverted to basal levels in LNCaP cells co-treated with 100 µM bicalutamide and 1 nM R1881. In contrast, KLK14 expression was down-regulated by R1881, derepressed with the addition of 10 and 50 µM bicalutamide, and stimulated with 100 µM bicalutamide (Figure 3.14B, P<0.001). These data, together with the hormone deprivation experiments, confirm the differential androgen regulation of prostatic kallikreins.

3.3.5 Androgen Receptor Knock Down Alters Prostatic Kallikrein Expression The expression of KLK2, 3, 4, 14 and 15 is sensitive to the addition and withdrawal of androgens. AR levels were attenuated with siRNA in LNCaP cells to determine whether the AR has a direct role in modulating kallikrein expression. A significant decrease in AR mRNA and protein levels was observed in LNCaP cells transfected with AR siRNA compared with non- targeting control oligonucleotides (Figure 3.15A & B). Although AR protein levels were stabilised by androgens, there was a similar relative decrease of AR in LNCaP cells treated with ethanol and R1881. Reduced KLK3 production in cells transfected with AR siRNA and treated with R1881 confirmed the loss of functional AR. In the presence of R1881, KLK2, 3, 4 and 15 expression was significantly reduced in LNCaP cells transfected with AR siRNA versus the non- targeting control (Figure 3.15C; KLK2, 4, 15 P<0.05, KLK3 P<0.01). In comparison, relative KLK14 levels were significantly increased with AR knock down due to decreased androgen- mediated repression (P<0.05). This change was too subtle to detect with Western blots (Figure 3.15A). The already low basal levels of KLK2, 3 and 4 in LNCaP cells treated with ethanol were further reduced with AR siRNA (Figure 3.15D). There was no change in KLK14 or KLK15 expression under these conditions.

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Figure 3.14. Bicalutamide Reverses R1881-Induced Changes in Kallikrein mRNA Levels. Charcoal-stripped LNCaP cells (48 hrs) were treated with 0 to 100 μM bicalutamide (BIC) for 2 hrs and then treated for an additional 24 hrs with 1 nM R1881 and the same concentration of bicalutamide. Changes in kallikrein expression were quantified using QRT-PCR and normalised to cells treated with R1881 only. Graphs represent average fold changes and SEMs (n=3, One Way ANOVA with Tukey’s posthoc analysis, # = KLK2: P<0.01, KLK3: P<0.05, KLK4: P<0.001, KLK15: not significant, ## = KLK2: P<0.001, KLK3: P<0.001, KLK4: P<0.001, KLK15: P<0.01, ### = P<0.001 for all genes, * P<0.05, ** P<0.01, ***P<0.001).

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(Figure 3.15, see over)

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Figure 3.15. Expression of Kallikreins in LNCaP Cells with AR Knock Down. LNCaP cells were cultured in 10% CSS containing media for 72 hrs, transfected with 100 nM control or AR siRNA for 48 hrs and then treated with 1 nM R1881 for 48 hrs. A - AR, KLK3 and KLK14 protein levels were quantified using Western blots with GAPDH as a loading control. A significant decrease in AR at the protein level was verified using densitometry comparing AR and GAPDH (n=3, t test, P<0.05). B – QRT-PCR demonstrating a significant decrease in AR mRNA levels in cells transfected with AR siRNA (n=3, t test, P<0.01). QRT- PCR was used to determine the expression of kallikreins in LNCaP cells transfected with control or AR siRNA and treated with 1 nM R1881 (C) or ethanol (D). Average fold changes compared with untransfected LNCaP cells are graphed with SEM (n=3, t test, *P<0.05, **P<0.001). E – A table of corresponding fold changes in kallikrein levels in response to R1881 and a scatterplot which highlights the negative correlation between kallikrein expression upon R1881 treatment (log scale) and after AR knockdown.

Notably, there appeared to be a relationship between the effects of AR knockdown and the sensitivity of each kallikrein to androgens (Figure 3.15E). For example, KLK2 and KLK3 have the largest fold changes with R1881 treatment and were down-regulated to the greatest extent with AR siRNA. These results demonstrate that the AR has an integral role in regulating the expression of prostatic kallikreins.

3.3.6 Androgens Do Not Alter the mRNA Stability of Prostatic Kallikreins Androgens regulate the abundance of target genes not only through direct AR-mediated transcriptional changes but also by altering mRNA stability. Indeed, the AR itself is post- transcriptionally androgen-regulated. AR transcription is down-regulated in the presence of androgens, yet AR protein levels increase because androgens increase AR mRNA stability (Yeap et al., 2004). The relative stability of KLK2, 3, 4, 14 and 15 mRNA was compared in LNCaP cells treated with ethanol or R1881. Transcription was inhibited with 20 µM actinomycin D and copy numbers of kallikrein transcripts remaining after 2, 4, 6 and 8 hours of treatment were measured using QRT-PCR. Interestingly, there were differences in mRNA stability between kallikreins: KLK14 degraded more readily than KLK2, 3, 4 and 15 (Figure 3.16). However, there was no change in the mRNA stability of prostatic kallikreins in LNCaP cells in the presence or absence of androgens. These results demonstrate that changes in kallikrein mRNA levels in response to androgens are not due to altered mRNA stability but rather direct transcriptional regulation of gene expression.

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Figure 3.16. Kallikrein mRNA Stability in LNCaP Cells Treated with Ethanol or R1881. LNCaP cells were cultured in 10% CSS for 72hrs and ethanol or R1881 (1 nM) for 24 hrs before 10 µg/mL actinomycin-D was added to stop transcription. Copy numbers of kallikrein transcripts in cells collected after 2, 4, 6 and 8 hours of treatment were measured using QRT-PCR and normalised to untreated cells (100%). The average percentage of transcripts remaining is plotted +/- SEM (n=3).

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3.3.7 Kallikrein Genes With Similar Expression Profiles are Clustered Together The synchronicity of changes in KLK2, 3, 4 and 15 expression in response to androgens is interesting given the adjacent localisation of these genes in the kallikrein locus. To further explore the relationship between androgen regulation and chromosome localisation, the androgen responsiveness of all kallikreins expressed in LNCaP and 22Rv1 cells was measured and graphed according to the order of genes in the kallikrein locus. A centromeric cluster of genes that were all up-regulated by R1881 was observed in LNCaP cells (Figure 3.17A). KLK2 and KLK3, which have inverse direction of transcription to other kallikreins, were at the centre of this cluster and were the most highly up-regulated by androgens. The kallikrein-like KLKP1 (KRIP1) gene located between KLK2 and KLK4 was also up-regulated by R1881 treatment. KLK1 and KLK5 were modestly androgen regulated but lowly expressed in LNCaP cells. KLK11, which is located telomeric to KLK5, was also slightly androgen regulated but poorly expressed. KLK14, which was down-regulated by androgens, lies at the far telomeric end of the kallikrein locus. KLK6-10, 12 and 13 were not detected in LNCaP cells. A similar cluster of androgen regulated genes was apparent with 22Rv1 cells (Figure 3.17B), although KLK5 levels were below the detection limit. KLK11 is more highly expressed in 22Rv1 compared with LNCaP cells and was once again moderately stimulated by R1881. KLK10, which is adjacent to KLK11, was also up-regulated. KLK8 was down-regulated in addition to KLK14. There was little change in KLK13 expression. Significantly, these results have been independently replicated. Analysis of recently published microarray data reveals the same trend of KLK2, 3, 4 and 15 expression in DHT treated C42B cells which are a subline of LNCaP cells (Figure 3.17C; Jia et al., 2008). Therefore, the centromeric cluster of kallikrein genes is androgen regulated in LNCaP, 22Rv1 and C42B cells.

To identify other co-expression patterns, particularly among non-androgen regulated genes, the expression profile of all 15 kallikreins and KRIP1 was determined using RT-PCR with seven prostate cell lines (Figure 3.18). All genes were expressed in at least one cell line. Confirming their status as androgen regulated genes, expression of KLK1-4 and 15 as well as KLKP1 (KRIP1) was restricted to androgen responsive LNCaP, 22Rv1 and MDA-PCa-2b cell lines. KLK5 and KLK6 genes, which are adjacent in the kallikrein locus, were co-expressed in PC3 cells. Lower levels of KLK5 were also detected in other cell lines. KLK7-10 were most highly expressed in RWPE-1 and RWPE-2 cells but were also co-expressed to some degree in 22Rv1 and PC3 cells. KLK11 and KLK13 had a similar expression profile that was not shared by KLK12. KLK14 was the only kallikrein expressed in all prostate cell lines. These data suggest there are at least three clusters of kallikreins that are co-expressed in different subsets of prostate cancer cells; KLK1-4 and KLK15, KLK5 and KLK6, and KLK7-10.

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Figure 3.17. Androgen Regulation of Kallikrein Expression in LNCaP and 22Rv1 Cells Compared with Genomic Localisation. A&B - Changes in kallikrein expression in LNCaP and 22Rv1 cells treated with 1 nM R1881 for 24 hrs were calculated using QRT-PCR and normalised to cells treated with ethanol. Average fold changes and SEM are graphed according to each gene’s position in the kallikrein locus. Missing values indicate undetectable expression. C - Fold changes in kallikrein expression in C42B cells were calculated from supplementary data published by Jia and colleagues (2008). Average log2 fluorescence values from cells treated with 10 nM DHT for 16 hrs were normalised to ethanol controls from Illumina microarrays performed in triplicate. The direction of transcription for each gene is shown below each graph. For the LNCaP and 22Rv1 experiments, light blue arrows indicate detectable levels of gene expression and dark blue arrow undetectable gene expression. For the C42B data, light green arrows denote genes with one microarray probe, whereas dark green arrows indicate genes with two probes (KLK3, 10, 11).

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Figure 3.18. Expression of all 15 Kallikrein Genes in Prostate Cell Lines. RT-PCR (40 cycles) was used to the whether each kallikrein and the KLKP1 (KRIP1) gene are expressed in RWPE-1, RWPE-2, LNCaP, 22Rv1, PC3, DU145 and MDA-PCa-2b prostate cell lines.. Results are shown in the order of genes in the kallikrein locus. 18S was used as a control to compare cDNA concentrations. The potential luminal and basal groups of kallikreins are highlighted.

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RWPE-1, RWPE-2, LNCaP, 22Rv1, PC3, DU145 and MDA-PCa-2b cells were further characterised to determine whether the expression of each cluster of kallikreins is related to a particular cell type. The different stages of prostate epithelial cellular differentiation are defined with a range of phenotypic markers such CK5, 8, 14 and 18, p63, PSCA and AR (see Chapter 1.6). The dedifferentiation of aggressive prostate cancer cells during cancer progression is more complex than stepwise reversion through the normal stages of differentiation .Instead, prostate tumour cells can transdifferentiate and adopt the characteristics of other cellular lineages. For example, prostate cancer cells become more fibroblast-like and down-regulate epithelial markers including E-cadherin and begin to express mesechymal proteins such as N-cadherin and vimentin (Lawrence et al., 2007). Neuroendocrine transdifferentiation is another form of tumour plasticity. As previously noted, these changes can be monitored with neuron-specific enolase expression, although this is also a marker of bone fide neuroendocrine cells in the prostate (Yuan et al., 2007).

Based on the expression of phenotypic markers, the androgen regulated kallikrein cluster, KLK1-4 and KLK15, was restricted to androgen responsive luminal epithelial cell lines, LNCaP, 22Rv1 and MDA-PCa-2b (Figure 3.19). These cell lines all express cytokeratin 8 and 18, AR and KLK3. Interestingly, the MDA-PCa-2b cell line also expresses cytokeratin 14 and PSCA, implying that it has a subpopulation of intermediate cells. The cytokeratin profile of DU145 and PC3 cells suggests that they have a luminal phenotype; however they do not express KLK1-4 and KLK15 because they lack AR. These results correlate with the glandular localisation of KLK2, 3, 4 and 15 in immunohistochemistry. KLK5 and KLK6 expression, which was highest in PC3 cells, did not correlate with a particular prostate cell type. KLK7-10 were predominantly expressed in RWPE-1 and RWPE-2, cells which have a transit amplifying or intermediate phenotype given that they co-express cytokeratin 5, 8, 14 and 18, p63 and PSCA. Interestingly, KLK5-10 expression also correlates with ER status as ERα and ERβ are only expressed in RWPE-1, RWPE-2 and PC3 cells (Cheung et al., 2005). E-cadherin, N-cadherin, vimentin and NSE were not associated with kallikrein expression. CD133 expression (data not shown) was not observed, either suggesting that no prostate stem cells were present, or that the subpopulation was too small to detect with this technique. Nevertheless, these data indicate that separate clusters of kallikreins are expressed in different cell types. KLK1-4 and 15 are restricted to AR- positive luminal epithelial cells while KLK7-10 expression correlates with ER status as well as a transit amplifying or intermediate cell phenotype.

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Figure 3.19. Profiles of Phenotypic and Differentiation Markers in Prostate Cell Lines. RT-PCR was used to test the expression of cytokeratin 5, 8, 14 and 18 (CK5 etc), p63, prostate stem cell antigen (PSCA), AR, vimentin (VIM), N-cadherin (NCAD), E-cadherin (ECAD) and neuron-specific enolase (NSE) in RWPE-1, RWPE-2, LNCaP, 22Rv1, PC-3, DU145 and MDA- PCa-2b prostate cell lines.

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3.4 Discussion

3.4.1 Kallikreins are Differentially Androgen Regulated and Expressed in Clusters Individual members of the kallikrein family have been associated with prostate cancer as biomarkers and functional mediators of tumour progression (Stephan et al., 2007). Although it is likely that multiple kallikreins may be useful adjunct biomarkers and act together in enzymes cascades, little is known about their comparative expression profiles. This study extends previous research by establishing an integrated model of kallikrein expression and regulation in prostate cancer.

The expression of KLK2, 3, 4, 11, 14 and 15, the most highly expressed kallikreins in the prostate, was initially investigated using QRT-PCR of seven prostate cell lines with a spectrum of phenotypes. Copy numbers were calculated to ensure quantitative comparisons could be made between kallikreins, not just cell lines, as with previous studies (Harvey et al., 2000, Shaw and Diamandis 2007). KLK2, 3, 4 and 15 expression significantly correlated with AR status, while KLK14 was ubiquitously expressed among cell lines. KLK11 was poorly expressed in the prostate cell lines, in contrast to the observations of a previous study (Nakamura et al., 2003a) and KLK11 protein levels in prostate tissue extracts (Shaw and Diamandis 2007). The karyotypes of the cell lines used in these experiments should be noted: RWPE-1, RWPE-2 and MDA-PCa-2b cells are diploid, PC-3 and DU145 cells are triploid, and LNCaP cells are tetraploid (America Type Culture Collection and (van Bokhoven et al., 2003a)). 22Rv1 cells are mostly diploid at the low passage numbers used in this study but are tetraploid at higher passages (van Bokhoven et al., 2003a). Importantly, no alterations affecting the kallikrein locus on chromosome 19q13 have been reported in these cell lines (Ohnuki et al., 1980, Nupponen et al., 1998, van Bokhoven et al., 2003a). Therefore, for each cell line, the karyotype may influence the abundance of transcripts but the phenotype will determine whether each kallikrein is expressed or not.

The restricted expression profile of KLK2, 3, 4 and 15 to cell lines with a functional AR axis was an obvious indication that these kallikrein genes are highly androgen regulated. Indeed, the binding of AR to KLK2 and KLK3 promoters is well characterised (Kim and Coetzee 2004). In comparison, there are contradictory reports for KLK4, with observations of 2- to 40-fold up- regulation upon androgen treatment to no change at all (Nelson et al., 1999b, Yousef et al., 1999b, Korkmaz et al., 2001, Xi et al., 2004b, Dong et al., 2005a, Lazarevic et al., 2008). One semi-quantitative study indicated that KLK15 expression was stimulated by androgens but the magnitude of this response was unclear (Yousef et al., 2001c). Previous studies with prostate cancer cells have not compared the effect of androgens on more than two kallikreins at a time, so few quantitative comparisons can be made between genes. In this study, time and dose

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dependent increases in KLK2, 3, 4 and 15 expression were observed in LNCaP cells treated with R1881. These kallikreins were also up-regulated by R1881 in 22Rv1 cells. The fold changes were different for each kallikrein; however a consistent pattern was observed with KLK2, 3, 4 and 15 stimulated by R1881 with decreasing magnitudes. Fold changes in KLK2 and KLK3 expression were similar and approximately 10-fold greater than KLK4 and KLK15. The inverse changes in KLK2, 3, 4, and 15 expression observed with hormone deprivation, bicalutamide treatment and AR knockdown confirmed the integral role of the AR in regulating kallikrein expression in prostate cancer cells.

In contrast to other prostatic kallikreins, KLK14 expression was suppressed by androgens. The effect was modest but consistent with a 40 to 75% decrease depending upon the experiment. These changes are also specific because KLK14 levels increase with extended hormone deprivation, bicalutamide treatment and AR knockdown. These observations conform to the ubiquitous expression profile of KLK14 that is still expressed in androgen responsive cell lines. Therefore, KLK14 expression is modulated by androgens but does not display the almost on/off response of KLK2, 3, 4 and 15. This result was surprising because previous studies have indicated that KLK14 is up-regulated by androgens in ovarian and breast cancer cell lines, including T-47D (Borgono et al., 2003, Yousef et al., 2003a, Paliouras and Diamandis 2007, Paliouras and Diamandis 2008). Notably, KLK14 expression was down-regulated by hormone deprivation in T47D breast cancer cells but up-regulated in LNCaP prostate cancer cells (Figure 3.13B), implying that the kallikrein locus may be hormonally regulated in a cell specific manner. KLK6, 8, 10 and 13 are also up-regulated by androgens in breast but not prostate cancer cell lines (Paliouras and Diamandis 2007). Significantly, KLK6, 8, 10, 13 and 14 are all oestrogen regulated (Borgono et al., 2003, Paliouras and Diamandis 2007, Paliouras and Diamandis 2008). Therefore, another explanation for these differences is that the androgen used with the breast and ovarian cells, DHT, was being metabolised to 5α androstane-3β,17b-diol (3βAdiol) which binds to ERα and ERβ (Sikora et al., 2008). This indirect mechanism is supported by KLK14 up- regulation in DHT-treated Caov-3 (HTB-75) ovarian cancer cells which do not express AR (Lau et al., 1999, Yousef et al., 2003a). AR knockdown experiments in breast and ovarian cancer cell lines could be used to determine whether AR has a direct role in differentially regulating KLK14 expression in response to androgens.

Fold changes in kallikrein expression with androgen treatment vary between studies and between different experiments in this report. Conditions were optimised to maximise fold changes in this study so that otherwise moderate responses were not lost amongst experimental variation. Cells were cultured in media containing 10% CSS for 72 hrs prior to the addition of androgens. Some other reports have used 2% CSS for 24 to 48 hrs and have observed smaller fold changes (Dong

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et al., 2005a), perhaps due to the lower concentration of growth factors and other nutrients. The hormone deprivation experiment demonstrates that baseline kallikrein expression would also be higher after only 24 to 48 hrs of culture in CSS, decreasing the magnitude of fold changes when relative kallikrein expression was calculated. This also explains the larger fold changes measured in the AR knockdown experiment where cells were cultured in CSS for an extra 48 hrs while being transfected. The passage number of LNCaP cells also influences the magnitude of fold changes. Greater responses to androgens are observed in cells over 60 passages due to constitutive Akt activation (Lin et al., 2003). Passage 15 to 25 LNCaP cells were used in this study. The only exception is the R1881 dose response experiment, where passage 40 LNCaP cells were used, and which gave slightly higher fold changes. These observations emphasise the need to examine multiple kallikreins simultaneously because of the difficulties in making quantitative comparisons between different studies.

One of the most intriguing findings of this study was the difference in mRNA and protein expression of the KLK4 variants. KLK4-254 has the typical structure of kallikreins with pre- and pro-regions for secretion and proteolytic activation. KLK4-205, which cannot be secreted because it lacks these domains, has previously been observed to have nuclear localisation (Korkmaz et al., 2001, Xi et al., 2004b, Dong et al., 2005a). In agreement with other studies, the KLK4-254 transcript was expressed at much lower levels than the truncated KLK4-205 mRNA variant in LNCaP cells (Korkmaz et al., 2001, Myers and Clements 2001, Xi et al., 2004b, Dong et al., 2005b, Lai et al., 2009). Both transcripts were up-regulated by androgens to a similar degree. Unexpectedly, corresponding protein was detectable for the lowly expressed KLK4-254 variant but not the highly abundant KLK4-205 form. Although KLK4-254 was not observed in Western blots of whole cell lysates, it was successfully detected as an androgen-regulated 30-32 kDa doublet in LNCaP conditioned media, a less complex sample that is more amenable to concentration. Any bands for DU145 cells, which do not express KLK4 mRNA, were deemed to be non-specific. It is unlikely that the lack of KLK4-205 in Western blots was due to technical difficulties or unusual cellular packaging of endogenous KLK4-205, because transfected KLK4- 205 was readily detectable. Nuclear localisation of transfected KLK4-205 is commonly observed with immunofluorescence (Korkmaz et al., 2001, Xi et al., 2004b, Dong et al., 2005a). However, no nuclear staining of endogenous KLK4 was observed in immunofluorescence of LNCaP cells or immunohistochemistry of prostate tissue specimens. These results imply that KLK4-205 mRNA, which is of similar abundance and stability to KLK2 and KLK3 (Figures 3.1 & 3.16), is poorly translated. The 5’UTR of KLK4-205, which is different from classical kallikrein transcripts, may not support correct docking of RNA binding proteins and processing by ribosomes. Indeed, the native 5’ UTR is not included in KLK4-205 constructs that do produce protein when transfected into cells (Korkmaz et al., 2001, Xi et al., 2004b, Dong et al., 2005a).

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In contrast to KLK4-205, endogenous KLK4-254 is lowly expressed but is still translated and secreted into seminal plasma and cell line conditioned media (Obiezu et al., 2002, Dong et al., 2005a, Obiezu et al., 2005). Furthermore, in immunohistochemistry, KLK4-254 is observed in the cytoplasm of epithelial cells in prostate and other tissues (Dong et al., 2001, Day et al., 2002, Obiezu et al., 2002, Davidson et al., 2005, Dong et al., 2005a, Veveris-Lowe et al., 2005, Davidson et al., 2007, Gao et al., 2007, Ramsay et al., 2008a). These results demonstrate that KLK4-254 is the biologically relevant form of KLK4 in the prostate.

The observations in this study clearly vary from previous reports of KLK4-205 expression and may be attributable to differences in reagents. For example, a series of studies focussing on KLK4-205 have mistakenly used an anti-KLK4 antibody raised against a peptide (QIINGEDCSPHSQPW) 6 amino acids upstream of the KLK4-205 start codon (Xi et al., 2004b, Klokk et al., 2007). This antibody can only detect KLK4-254 and not KLK4-205 (Simmer and Bartlett 2004). The authors describe “predominantly nuclear” staining of prostate specimens in immunohistochemistry with this antibody, although published results have distinctly cytoplasmic localisation (Klokk et al., 2007). This antibody also yields cytoplasmic staining of breast and ovarian cancer samples, tissues that similarly co-express KLK4-254 and KLK4-205 mRNA (Xi et al., 2004a, Davidson et al., 2005). Western blots of KLK4-205 in these studies are also puzzling. They detect various 38-47 kDa bands which are approximately 10 kDa larger than the predicted molecular weight of KLK4-205 and yet are not glycosylated (Myers and Clements 2001, Xi et al., 2004b, Dong et al., 2005a, Gao et al., 2007, Klokk et al., 2007). These bands are also larger than transfected KLK4-254 and KLK4-205, recombinant human KLK4-254, purified porcine KLK4-254, and all other endogenous prostatic kallikreins, which are glycosylated in eukaryotic cells (Simmer et al., 1998, Takayama et al., 2001b, Obiezu et al., 2002, Ryu et al., 2002, Harvey et al., 2003, Xi et al., 2004b, Dong et al., 2005a, Matsumura et al., 2005, Obiezu et al., 2005, Beaufort et al., 2006, Debela et al., 2006, Obiezu et al., 2006, Klokk et al., 2007). Furthermore, the expression of these bands is frequently discordant with the KLK4-205 mRNA profile (Myers and Clements 2001, Dong et al., 2005a, Gao et al., 2007, Lazarevic et al., 2008). These experiments will remain contentious until the specificity of the reagents used to detect KLK4-205 is verified.

The co-ordinated up-regulation of KLK2, 3, 4 and 15 with androgens was of interest given the adjacent localisation of these genes in the kallikrein locus. To further investigate the relationship between androgen regulation and chromosome localisation, changes in the expression of all kallikreins expressed in LNCaP and 22Rv1 cells upon androgen treatment were measured. This experiment indicated that there is a hub of androgen-regulated genes at the centromeric end of the kallikrein locus centred on KLK2 and KLK3 and extending to KLK1 and KLKP1 (KRIP1).

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The expression of these kallikreins was restricted to cell lines with a luminal phenotype, in agreement with the high levels of these kallikreins detected in seminal plasma (Shaw and Diamandis 2007).

Another cluster of kallikreins, KLK7-10, was observed in RWPE-1 and RWPE-2 cells. There are only a few reports of KLK7-10 expression in the prostate. ELISAs of patient serum found no change in KLK8 or KLK10 levels, while RT-PCR analysis of 7 matched samples indicated that KLK10 was more highly expressed in benign specimens (Luo et al., 2001, Yousef et al., 2005, Parekh et al., 2007). Previous studies have examined the tissue expression profiles of kallikreins using common cell lines such as LNCaP, PC3 and DU145, and crude tissue extracts (Harvey et al., 2000, Shaw and Diamandis 2007). RWPE-1 and RPWE-2 cells have a transit amplifying and intermediate cell phenotype. Therefore, KLK7-10 expression in the prostate may have been overlooked because transit amplifying and intermediate cells only make up a small proportion of cells compared with luminal epithelial cells. However, KLK7-10 levels also mirror ERα and ERβ expression which is high in RWPE-1, RWPE-2 and PC-3 cells and low or absent in 22Rv1, DU145, LNCaP and MDa-PCa-2b cells (Cheung et al., 2005). Interestingly, Estrogen Receptor- Related Receptor β (ERRβ), an orphan receptor that is not activated by oestrogen, is also expressed in RWPE-1 and RWPE-2 cells (Cheung et al., 2005). KLK7-10 are all oestrogen- regulated (Yousef et al., 1999a, Yousef and Diamandis 2000, Yousef et al., 2000e, Katsu et al., 2002, Luo et al., 2003b, Yousef et al., 2003a, Paliouras and Diamandis 2007). In addition, KLK5 and KLK6, which were also expressed in PC-3 cells, are up-regulated by oestrogen (Paliouras and Diamandis 2007, Shaw and Diamandis 2008). In adult prostate tissue, ERβ and ERRβ are more highly expressed in luminal than basal epithelial cells and are down-regulated during carcinogenesis (Cheung et al., 2005, McPherson et al., 2008). Therefore, it is unclear whether KLK7-10 expression is associated with ER status or basal cells in vivo. These alternatives should be investigated with immunohistochemistry and in vitro oestrogen treatments. Nevertheless, by comparing kallikrein expression profiles this study has identified two clusters of co-expressed genes, KLK1, 2, 3, 4 and 15 and KLK7-10.

3.4.2 Potential Mechanism of Kallikrein Transcriptional Regulation The co-ordinated expression of groups of kallikrein genes raises interesting questions about their transcriptional regulation. Do kallikreins within each cluster have separate promoters with similar regulatory elements or are they jointly regulated by more broadly acting enhancer regions? For all kallikreins in the KLK1, 2, 3, 4 and 15 cluster to be distinctly androgen- regulated, each gene would require one or more AREs. To date, AREs have only been identified for KLK2 and KLK3. Two proximal promoter AREs, AREI (-156 to -170 bp) and AREII (-376 to -395 bp), have been characterised for KLK3, in addition to a potent enhancer ARE (AREIII; -

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4134 to -4148) surrounded by five weaker, non-consensus elements (Kim and Coetzee 2004). These AREs are conserved in the KLK2 promoter, reflecting the close evolutionary relationship between KLK2 and KLK3. A recent study isolated a region in the KLK4 promoter (-1005 bp) that indirectly interacts with AR; however this putative ARE was not sufficient to induce androgen-regulated reporter gene expression. A construct spanning 2.8 kilobases of the KLK4 promoter was similarly unresponsive to androgens. Two AREs have been identified in silico for KLKP1 (KRIP1) but have not been experimentally verified (Kaushal et al., 2008). Therefore, the task still remains to identify and validate AREs for KLK1, KLK4, KLK15 and KLKP1.

Recent technological and methodological advances have sparked investigations into AR- promoter interactions on a genome-wide scale. These studies suggest that novel AREs in the kallikrein locus may be challenging to identify with traditional approaches. Coupling chromatin immunoprecipitation (ChIP) experiments with PCR amplification and sequencing (ChIP Display) or oligonucleotide arrays (ChIP-Chip) has enabled identification of direct AR binding regions throughout the genome (Bolton et al., 2007, Jariwala et al., 2007, Massie et al., 2007, Prescott et al., 2007, Takayama et al., 2007, Wang et al., 2007). Most of these sites are greater than 10 kilobases from the genes they regulate and few are in proximal promoters (Bolton et al., 2007, Takayama et al., 2007, Wang et al., 2007). One study calculated that only 32% of AR binding regions are within 500 kilobases of transcription start sites of androgen-regulated genes (Wang et al., 2007). Many intragenic and 3’ AR binding regions have also been noted, including some that have been experimentally validated (Zheng et al., 2006, Bolton et al., 2007, Jariwala et al., 2007). Adding further complexity, few AREs in these AR binding regions (10 to 26.8%) conform to the 13 bp consensus sequence and instead consist of 6 base pair half sites (Massie et al., 2007, Wang et al., 2007). These findings suggest that it may be difficult to identify functional AREs for KLK1, 4, 15 and KLKP1 using transcription factor binding site prediction software and promoter deletion constructs. The AREs may be non-consensus and far removed. An alternative approach would be to mine ChIP-Chip data for novel AR binding regions near the kallikrein locus.

If AREs cannot be found for each gene in the androgen-regulated cluster of kallikreins, another possibility is that they are co-ordinately regulated by changes in chromatin conformation or a locus control region (LCR). KLK2 and KLK3 are strongly androgen regulated and are among the most highly expressed genes in prostate epithelial cells (Stanbrough et al., 2006). One hypothesis is that ligand-bound AR induces an open chromatin conformation necessary for high levels of KLK2 and KLK3 transcription that extends to neighbouring genes. This would allow greater access of basal transcription machinery to KLK1, 4, 15 and KLKP1 promoters, indirectly increasing their expression in response to androgens. However, a previous investigation noted

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that the widespread histone acetylation of KLK2 and KLK3 genes did not extend to the region between them (Jia et al., 2006). DNA torsional forces from chromatin unwinding and high levels of transcription can also trigger gene activation, although it is unknown how far along the chromosome these forces are transmitted (Kouzine et al., 2008).

Maintenance of open chromatin conformation is also characteristic of LCRs which enhance the tissue-specific expression of linked genes according to the number of enhancer elements (Li et al., 2002). The β-globin and human growth hormone LCRs are the best characterised of at least 21 human LCRs that have been studied (Li et al., 2002). No androgen induced LCRs have been identified, although other clusters of androgen-regulated genes similar to the kallikreins have been reported (Bolton et al., 2007). The existence of kallikrein LCRs has been posited in several studies to explain clusters of kallikreins with different tissue-specific and hormone-regulated expression profiles (Smith et al., 1992, MacDonald et al., 1996, Harvey et al., 2000, Yousef et al., 2003d, Paliouras and Diamandis 2007). Kroon and colleagues (1996) tested this theory with transgenic mice bearing genomic fragments of a rat-specific portion of the kallikrein locus. This region is comprised of 9 functional genes in an insertion between KLK2 and KLK4. The rat kallikreins were detected at physiological levels in corresponding mouse tissues, particularly the submandibular gland where they are usually co-expressed (Kroon et al., 1997). These findings implied that tissue-specific regulatory elements are proximal to rat kallikreins genes rather than at a distal LCR. Notably, the prostate was the only tissue where correct expression was not observed. KLK1c3 and KLK1c8 were highly expressed in rat prostate but were low or undetectable in the prostates of transgenic mice (Kroon et al., 1997). This indicates that these genes may have prostate-specific distal regulatory elements. The broader significance of these findings is not clear because human kallikrein genes have not been tested. Further studies are required to define the role of chromatin conformation in kallikrein expression and determine whether an androgen-responsive kallikrein LCR exists.

The tissue-specific and hormone-regulated expression of kallikreins in other mammalian species may provide clues about their transcriptional regulation in humans. If each kallikrein gene has its own set of regulatory elements, it is possible that they may be conserved throughout species. As mentioned, this is the case for KLK2 and KLK3, although only humans and primates have both genes. The duplication that gave rise to KLK2 and KLK3 included the promoter and enhancer AREs. These elements are also conserved in the canine KLK2-like gene, arginine esterase (Dube et al., 1995, Olsson et al., 2004, Lundwall et al., 2006). In contrast, KLK2 is a pseudogene and is not expressed in rodents and the cotton top tamarin because AREI and AREIII have been inactivated by 3-15 base pair deletions (Olsson et al., 2005). These observations stress the importance of the AREs for correct KLK2 and KLK3 expression.

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KLK1 is also androgen-regulated in multiple species. It is expressed in the porcine prostate and up-regulated by androgens in the rat submandibular gland (Chao and Margolius 1983, Miller et al., 1984, Gerald et al., 1986, Fernando et al., 2007). KLK1 has been duplicated multiple times in the mouse genome and some of these paralogs are androgen regulated (van Leeuwen et al., 1987, Eacker et al., 2007). The androgen responsive region has been isolated to the proximal promoter for one of these mouse genes, KLK1b27 (Eacker et al., 2007). In the rat genome, KLK1, 2 and 15 were tandemly duplicated in a 430 kilobase region between KLK2 and KLK4 (Olsson et al., 2004). All KLK1 parologs that are in same orientation as KLK1 (KLK1c2, KLK1c3, KLK1c8 and KLK1c9) are androgen-regulated and expressed in the rat prostate (Ashley and MacDonald 1985, Shih et al., 1986, Clements et al., 1988, MacDonald et al., 1996). Therefore, like KLK2 and KLK3, KLK1 may have evolutionarily conserved androgen- responsive regions. Notably, KLK1, 2 and 3 arose most recently and share the highest homology among kallikreins (Olsson et al., 2004). There is no evidence for conserved elements that drive the prostatic expression of KLK4 and KLK15 in human as neiher kallikrein is expressed in the porcine prostate (Fernando et al., 2007). These observations suggest a mixed model for regulation of the prostatic cluster of kallikreins; direct, gene-specific regulation of KLK1, 2 and 3 and indirect induction of KLK4 and KLK15. With this model it is possible that the KLK2 and KLK3 enhancers act like LCRs for KLK4 and KLK15. Chromatin conformation capture assays have shown that the KLK3 proximal promoter loops to its enhancer region (Wang et al., 2005). Similar experiments could be conducted to test for interactions between the KLK2 or KLK3 enhancer and the KLK4 and KLK15 proximal promoters.

It is likely that KLK14 is regulated independently of other kallikrein genes in the prostate. KLK14 lies at the extreme telomeric end of the kallikrein locus and has a unique expression profile in prostate cell lines. KLK14 is also separated from the androgen-regulated group of kallikreins by the oestrogen-regulated or basally expressed KLK7-10 cluster, which may be demarcated by insulator regions itself. Furthermore, unlike KLK1-4, KLK14 is down-regulated by androgens. Groups of androgen-regulated genes are usually all induced, such as the ephrin cluster, or repressed by androgens, such as the CXC chemokine locus (Bolton et al., 2007). The repressive actions of AR are well recognised but poorly understood. Studies have noted that slightly fewer genes are repressed by androgens than stimulated (Wang et al., 1997, Clegg et al., 2002, Jiang and Wang 2003, Martin et al., 2004, Bolton et al., 2007, Prescott et al., 2007). Like KLK14, most of these genes are moderately down-regulated rather than completely repressed (Kojima et al., 2006, Prescott et al., 2007). One potential mechanism is that AR actively inhibits target gene expression by recruiting co-repressor proteins such as NCoR and SMRT. This is unlikely for KLK14 and some other genes because their expression is rescued by bicalutamide which maintains the co-repressor interactions and DNA binding ability of AR (Kojima et al.,

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2006, Hodgson et al., 2007, Prescott et al., 2007). AR also down-regulates expression without binding to target gene promoters. It can compete for limited pools of co-activators or form inhibitory interactions with other transcription factors such as NFκB, Sp1, AP-1, cJun and ATF2 (Palvimo et al., 1996, Schneikert et al., 1996, Curtin et al., 2001, Jorgensen and Nilson 2001a, Jorgensen and Nilson 2001b, Verras et al., 2007). Therefore, the KLK14 promoter should be further characterised to help determine the mechanism of AR-mediated repression.

3.4.3 The Clinical Implications of Kallikrein Expression in Prostate Cancer. Previous reports have suggested that particular kallikreins are differentially expressed in prostate cancer and have functional roles in tumour progression. However, the expression profiles and proteolytic functions of different kallikreins are likely to be interconnected. In this study an integrated model of kallikrein expression and regulation at the cellular level was established. The next step will be to apply these findings to disease progression in patients.

Androgen-regulated genes like the kallikreins are generally down-regulated as the primary tumour dedifferentiates and are then further reduced in metastases (Hendriksen et al., 2006). In those patients that undergo chemical or surgical castration, androgen-regulated genes are down- regulated but are then re-expressed in hormone-refractory disease (Holzbeierlein et al., 2004). Although KLK3 expression follows these trends, it is highly heterogeneous after castration as tumours proceed towards androgen independence (Mostaghel et al., 2007). This variability may be due to the numerous ways that prostate cancer cells adapt to castrate androgen levels including over-expression and mutation of AR, up-regulation of transcriptional cofactors, and increased intratumoural production of androgens (Schroder 2008). Under these conditions subsets of androgen-responsive genes may also be differentially regulated. For example, KLK2 and KLK3 expression does not correlate with the androgen-induced growth of prostate cancer cells (Denmeade et al., 2003b). Therefore, prostatic kallikrein expression in patients is likely to be heterogeneous. Unlike their cell line expression profile, androgen-regulated kallikreins will be maintained in hormone refractory prostate cancer, albeit at lower levels than in androgen responsive tumours.

The coordinated androgen-regulation of KLK1, 2, 3, 4 and 15 implies that they have similar expression patterns in prostate cancer. However, previous studies suggest that KLK3 is decreased in cancer versus benign specimens, whereas KLK4 and KLK15 are increased (Stephan et al., 2007). KLK1 expression has been noted in patient specimens but not examined in disease progression (Clements and Mukhtar 1997). These observations might suggest that KLK4 and KLK15 expression becomes uncoupled from KLK2 and KLK3 in prostate cancer progression. KLK4 and KLK15 have broader tissue expression profiles so it is possible that

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some of the factors influencing their extra-prostatic expression are up-regulated in prostate cancer. However, it is difficult to compare the results of previous studies because each kallikrein has been tested with different cohorts and methodologies. Some of these studies are also confounded by technical limitations such as small numbers of specimens. The specificity of some antibodies used for immunohistochemistry, particularly for KLK4, has also been challenged (Simmer and Bartlett 2004, Obiezu et al., 2005). In addition, RT-PCR and QRT-PCR are often used to compare kallikrein levels in patient tissue extracts with unknown proportions of normal to malignant cells, epithelial to stromal cells, and different grades of cancer. Conflicting reports of KLK3 expression exemplify this problem. Increased KLK3 levels are detected in crude extracts of cancer versus benign specimens, whereas a decrease is observed with more targeted techniques such as immunohistochemistry, in situ hybdridisation, and QRT-PCR of laser capture microdissected samples (Qiu et al., 1990, Yang et al., 1992, Hakalahti et al., 1993, Culkin et al., 1995, Grande et al., 2000, Kaushal et al., 2008, Sterbis et al., 2008). It is important to note that reports of increased KLK15 in prostate cancer are solely based on mRNA levels in crude extracts (Yousef et al., 2001c, Stephan et al., 2003, Michael et al., 2005). Therefore, the expression profiles of KLK4 and KLK15 in prostate cancer require further clarification.

In this study, immunohistochemistry was used to compare the expression of several kallikreins in serial sections of prostate tissue. Multiple primary antibodies were used for KLK4, 14 and 15 because their expression pattern is less well characterised. Most of these antibodies have been tested for cross-reactivity against other kallikreins: however their specificity should be confirmed using negative control tissues that lack kallikrein expression. The preliminary data showed that the prostatic kallikreins were co-expressed. A different trend in KLK4 expression was observed compared with previous studies. Like KLK3, KLK4 levels were lower in cancer compared with adjacent benign regions. Clearly, greater numbers of specimens must be tested to gain an accurate expression profile and overcome the heterogeneity between patients. Although they are quite rare, it will be important to include samples from men undergoing androgen-deprivation therapy and with hormone refractory prostate cancer in these analyses.

It will be particularly interesting to investigate KLK14 expression in more detail, since it is down-regulated by androgens. Previous studies have shown that KLK14 is up-regulated in cancer compared with benign prostate specimens (Yousef et al., 2003e, Rabien et al., 2008). Preliminary immunohistochemistry conducted in this study supports these observations. Although the effect of castration on KLK14 expression has not yet been reported, other genes that are repressed by androgens, such as IGFBP3 and relaxin, are initially up-regulated by hormone deprivation therapy but then decrease in androgen-independent stages of prostate cancer (Kojima et al., 2006, Thompson et al., 2006). Therefore, the expression profile of KLK14

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may be opposite to KLK3. If this is the case, the ratio of KLK3 to KLK14 expression may be a useful measure of AR activity that overcomes some of the variability between patients. Further analysis of the expression profiles of KLK3, KLK14, and other prostatic kallikreins is warranted to test this possibility.

In summary, the research presented in this chapter is the most comprehensive analysis of kallikrein expression in prostate cancer to date. Multiple kallikreins were examined in each experiment so that quantitative comparisons could be made, in particular between their androgen responsiveness. Intriguingly, many kallikreins were expressed in clusters, emphasising that their functions in cancer progression are likely to be interconnected. In future studies these findings should be expanded to larger cohorts of patient specimens spanning different stages of prostate cancer progression, including castration and hormone refractory disease.

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4Chapter 4: The Role of β-catenin in Kallikrein-Related Serine Peptidase Expression

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4.1 Introduction The progression of prostate cancer is characterised by increasing tumour dedifferentiation (Marker 2008). Highly aggressive prostate cancer cells re-express developmental factors, including Wnts, and acquire a more plastic phenotype. For example, epithelial to mesenchymal transition, with characteristic loss of E-cadherin, provides prostate cancer cells with a selective advantage to invade (Whitbread et al., 2006). Many of these pathways antagonise AR signaling which typically promotes differentiation and is vital for the growth and survival of prostate cancer cells (Lawrence et al., 2007, Balk and Knudsen 2008). Therefore, prostate cancer cells must strike a balance between the dedifferentiated phenoytype and the AR axis. The multifunctional signaling molecule β-catenin connects these opposing pathways and others and may promote the progression of prostate cancer (Cheshire and Isaacs 2003).

β-catenin exists in three distinct pools, the cell adhesion complex, cytoplasm and nucleus, which determine its function and how it is regulated. β-catenin is a key component of adherens junctions in normal prostate epithelial cells, stabilising cell-cell contacts by linking E-cadherin to the actin cytoskeleton via α-catenin (Mason et al., 2002). To enhance cellular migration during embryonic development or cancer progression, the cell adhesion complex is disrupted through down-regulation of E-cadherin or phosphorylation of β-catenin by tyrosine kinases such as Src, c-met and EGFR (Lilien and Balsamo 2005). β-catenin subsequently accumulates in the cytoplasm where it is sequestered by the multiprotein degradation complex (Lustig and Behrens 2003). GSK3β, which is active in prostate epithelial cells under basal conditions, phosphorylates β-catenin, triggering its ubiquitination and proteosomal degradation (Doble and Woodgett 2003). Wnt ligands alleviate β-catenin degradation through the canonical pathway by inactivating GSK3β (Lustig and Behrens 2003). This cytoplasmic pool of β-catenin can then shuttle to the nucleus, bind to the TCF family of transcription factors, predominantly TCF4 in the prostate, displace corepressors, recruit coactivators, and induce expression of target genes.

Of particular relevance to prostate cancer, β-catenin not only binds to TCF, but also AR (Truica et al., 2000, Mulholland et al., 2002, Pawlowski et al., 2002, Yang et al., 2002, Song et al., 2003, Wang et al., 2008b). This interaction is agonist-dependent, facilitates the nuclear translocation of β-catenin, and results in more potent stimulation of androgen responsive genes (Truica et al., 2000, Chesire et al., 2002, Mulholland et al., 2002, Yang et al., 2002, Song et al., 2003, Masiello et al., 2004). Significantly, AR and TCF activities are mutually exclusive, presumably due to competition for β-catenin and other shared co-activators. Over-expression of either transcription factor in vitro inhibits the transcriptional activity of the other (Chesire et al., 2002, Chesire and Isaacs 2002, Pawlowski et al., 2002, Amir et al., 2003, Mulholland et al.,

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2003, Verras et al., 2004). For example, TCF activity increases during neuroendocrine transdifferentiation after prolonged androgen deprivation (Yang et al., 2005b). In an in vivo rat model, prostatic TCF activity was highest during development when androgen concentrations are low, undetectable in adulthood, reactivated upon castration and then inhibited when androgens were re-administered (Wang et al., 2008a). Therefore, in prostate cancer the actions of β-catenin depend on cellular context; the expression of the TCF and AR axes, and the levels of androgens.

As noted in the previous chapter, several kallikrein-related serine peptidases are androgen- regulated. Therefore, it is likely that their expression levels will be influenced by the AR:β- catenin interaction. Indeed, much is already known about the role of β-catenin in KLK3 expression since it is commonly used as a measure of AR activity. Over-expression of wild-type or GSK3β-resistant mutant β-catenin increases ligand-dependent AR activation of KLK3 promoter constructs in several cell lines (Yang et al., 2002, Amir et al., 2003, Li et al., 2004, Verras et al., 2004, Chen et al., 2006b). Moreover, ChIP assays have shown that β-catenin is recruited by AR to the -170 ARE in the KLK3 proximal promoter in LNCaP cells (Amir et al., 2003, Li et al., 2004, Liu et al., 2008b). Similar to KLK3, the activity of a KLK2 reporter construct was stimulated by β-catenin transfection above the levels of ligand-bound AR alone (Chesire et al., 2002). Whether β-catenin regulates other prostatic kallikreins is unknown. Given that KLK4 and KLK15 are also up-regulated by androgens, it is likely that β-catenin will enhance their expression in synergy with AR. Unlike other prostatic kallikreins, KLK14 is suppressed by AR. Therefore, it is possible that KLK14 expression is instead stimulated by β- catenin and TCF. In this situation, AR would indirectly inhibit KLK14 expression through competition for β-catenin. This hypothesis is supported by the up-regulation of KLK14 during neuroendocrine transdifferentiation in response to prolonged androgen deprivation when β- catenin levels and TCF activity are high in prostate cancer cells. In addition, KLK14 expression is rescued by bicalutamide which does not support AR binding to β-catenin and thus its sequestration from TCF (Masiello et al., 2004, Song et al., 2004).

The competition of the AR and TCF axes for β-catenin, an important signaling molecule in prostate cancer progression, may underlie the differential regulation of prostatic kallikreins by androgens. Therefore, the role of β-catenin in kallikrein expression warranted further investigation. Here, different models were used to assess the effect of the β-catenin:TCF and β- catenin:AR interactions. The results demonstrated that β-catenin is indeed important for the optimal expression of several prostatic kallikreins.

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4.2 Materials and Methods

4.2.1 Cell Culture LNCaP, DU145 and 22Rv1 cells were routinely cultured as previously outlined (Chapter 2.4). The DU145 sublines, DU145-E (epithelial-like) and DU145-F (fibroblast-like), and parental cells, DU145-P, were obtained from Elisabeth Seftor and Professor Mary Hendrix, Children’s Memorial Research Centre, Northwestern University Feinberg School of Medicine, Chicago, USA (Chunthapong et al., 2004). These cells and the human embryonic kidney cell line, 293T, were cultured in DMEM containing 10% FCS, 50 U/mL Penicillin G and 50 μg/mL Streptomycin (Invitrogen). LNCaP cell lines stably transfected with β-catenin siRNA in the pTER plasmid or vector only control were provided by Dr Shao-Yong Chen and Associate Professor Steven Balk, Beth Israel Deaconess Medical Centre, Harvard School of Medicine, Boston, USA (Masiello et al., 2004). The LNCaP cell lines were cultured in RPMI 1640 media with 10% fetal bovine serum, 50 U/mL Penicillin G, 50 μg/mL Streptomycin, 300 µg/mL G418 and 300 µg/mL zeocin (Invitrogen).

4.3 Inhibition of GSK3β Activity Cells were treated with 40 mM LiCl or KCl salt control (Sigma) for 24 hrs. SB216763, a more specific GSK3β inhibitor, was added to cells at 5 or 20 µM concentrations for 8 hrs, except for luciferase assays where 10 µM was added for 24 hrs. DMSO was used as vehicle control. Unless otherwise indicated, 22Rv1 cells were grown in CSS containing media for LiCl and SB216763 treatments.

4.3.1 Luciferase Assays The pGL3-OT (pOT) and pGL3-OF (pOF) reporter constructs were provided by Dr Bert Vogelstein, Howard Hughes Medical Institute and Sidney Kimmel Comprehensive Cancer Centre, Johns Hopkins University, Baltimore, USA (Shih et al., 2000). The pOT plasmid consists of three copies of the wild type TCF-4 binding element cloned into the pGL3-Basic vector (Promega) whereas pOF has three mutant binding sites and acts as a background control. The KLK3 reporter construct (pGL3-KLK3) contains approximately 5.8 Kb of the KLK3 promoter including all AREs (Lai et al., 2007). The human TCF-4 expression construct (pCMV- TCF-4) was from Dr Sergei Sokol, Beth Israel Deaconess Medical Centre, Harvard School of Medicine, Boston, USA (Korinek et al., 1997) while the wild type β-catenin expression construct (β-catenin pcDNA3.1) was provided by Dr Hans Clevers, The Hubrecht Laboratory, The Netherlands. For luciferase assays, LNCaP and 293T cells were grown in 48-well plates. For each well, 50 ng of pOT or pOF and 2.5 ng of pCMV-Renilla were transfected with 1 μL

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Lipofectamine 2000 using the standard protocol (Chapter 2.13). For some wells, 20 ng of β- catenin-pcDNA3.1 was also added. β-catenin was co-transfected with 20 ng of pCMV-TCF-4 for LNCaP cells. The media was changed 24 hrs after transfection and after an additional 24 hrs, cells were lysed with 50 uL passive lysis buffer (Promega). Luciferase activity was measured using a PolarStar plate reader using 20 uL of lysate with 40 uL LarII and Stop and Glow (Promega) per well. DU145 and 22Rv1 cells were cultured in 24-well plates for luciferase assays. For each well, 500 ng of pOT, pOF, pGL3-KLK3 or pGL3 Basic and 300 ng of phRL- TK Renilla (Promega) were transfected with 3 μL Lipofectamine 2000. Cells were transfected for 24 hrs, treated with LiCl or SB216763 for a further 24 hrs, and then lysed with 100 µL of passive lysis buffer. Luciferase activity was measured using 20 µL of lysate with 100 µL LarII and Stop and Glow.

4.3.2 Androgen Treatments The LNCaP β-catenin knockout and vector control cell lines were cultured in RPM1 containing 10% CSS for 72 hrs and then treated with 1 nM R1881 or ethanol control for 24 hrs.

4.3.3 QRT-PCR Total RNA was extracted using TRIzol, DNase I treated and then reverse transcribed with SuperScript III as previously described (Chapters 2.5 & 2.6). QRT-PCR was conducted using Sybr Green (Applied Biosystems) and primers listed in Appendix C using the cycling parameters outlined previously (Chapter 2.5).

4.3.4 β-catenin Knockout DU145 cells were seeded at 1 x 105 cells per well in 6-well plates and cultured for 48 hrs so that they reached approximately 30% confluency. Cells were washed with PBS and incubated in 800 µL of serum-free media prior to transfection. For each well, 100 pmol of four pooled β-catenin siRNAs or a non-targeting control siRNA (Dharmacon, Millenium Scientific, Surrey Hills, Vic, Australia) (5 µL of 20 pmol/L solution) was mixed with 180 µL OptiMEM for 5 mins. Another 11 µL of OptiMEM was separately mixed with 4 µL of Oligofectamine. The solutions were mixed, incubated for 20 mins at room temperature and then applied to the cells. After 4 hrs of transfection, 500 µL of RPMI 1640 containing 30% serum was added to each well to restore the serum concentration to 10%. Three days later, the cells were treated with 20 µM SB216763 or DMSO control for 8 hrs before RNA and protein extractions.

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4.3.5 Protein Extraction and Western Blotting Whole cell lysates were prepared as previously described (Chapter 2.10). To extract the soluble fraction, which largely excludes membrane-bound proteins, cells were mixed with buffer containing 10 mM PIPES pH 6.8, 300 mM sucrose, 5 mM EDTA, 3 mM MgCl2, 500 mM NaCl, 0.01% digitonin and 1x protease inhibitor (Sigma) and incubated on ice for 10 mins as previously reported (Ramsby et al., 1994). The lysates were then centrifuged at 4000 g for 10 min at 4 ºC to pellet insoluble protein and the supernatants were stored at -80 ºC. Western blots were conducted according to Chapter 2.11 using primary antibodies listed in Appendix D.

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4.4 Results

4.4.1 β-catenin is Required for Optimal Expression of Androgen-Regulated Kallikreins Being androgen-regulated genes, it is likely that KLK2, 3, 4 and 15 expression is altered by β- catenin binding to AR. Several studies have shown that LNCaP cells do not exhibit β- catenin:TCF activity (Chesire and Isaacs 2002, Chesire et al., 2004, Cronauer et al., 2005). This makes them a useful model to specifically investigate the effects of the β-catenin:AR interaction. Consistent with these reports, the activity of a TCF-responsive promoter construct (pOT) in LNCaP cells was less than the negative control construct (pOF), even with exogenous β-catenin and TCF-4 expression (Figure 4.1A). In contrast, 293-T cells, which were used as a positive control, had greater pOT activity in basal conditions and with β-catenin transfection. The role of β-catenin in androgen-regulated kallikrein expression was investigated using LNCaP cells stably transfected with shRNA targeting β-catenin or vector control (Masiello et al., 2004). β-catenin levels were significantly lower in the β-catenin knockout cell line (Figure 4.1B). In addition, a previous study demonstrated that there was little soluble, that is cytoplasmic and nuclear, β- catenin protein in these cells (Masiello et al., 2004). The β-catenin knockout and vector cell lines were cultured in CSS for 72 hrs and treated with 1 nM R1881 for 24 hrs before kallikrein expression was measured using QRT-PCR. There was a significant decrease in KLK2, 3 and 4 up-regulation in response to androgens in the β-catenin knockout cells (Figure 4.1C). For example, in the vector cell line KLK2 expression was 21.9-fold greater upon R1881 treatment compared to the ethanol control but only 8.6-fold increased in β-catenin knockout cells. KLK15 followed a similar trend but the difference between cell lines was not significant. There was also no significant difference in KLK14 expression which was once again down-regulated by R1881 treatment. These results demonstrate that β-catenin is required for maximal androgen stimulation of KLK2, 3 and 4 expression.

4.4.2 GSK3β Inhibition Increases KLK14 Expression A potential explanation for KLK14 down-regulation by androgens is that it is stimulated by β- catenin and TCF but then repressed when β-catenin is sequestered by ligand-bound AR. To test the hypothesis that KLK14 is regulated by β-catenin and TCF, DU145 and 22Rv1 cells were used because they are known to have a functional TCF axis (Chesire and Isaacs 2002, Chesire et al., 2004). Cells were treated with 20mM LiCl to inhibit GSK3β activity and therefore β-catenin degradation. KCl was used as a negative control. 22Rv1 cells were treated in CSS containing media to suppress AR activity in favour of TCF. This was not necessary for DU145 cells which lack AR. TCF activity measured with the pOT reporter construct in both of these cell lines was

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Figure 4.1. Kallikrein Expression in β-catenin Knockout LNCaP Cells Treated with R1881.

A – Luciferase assays with LNCaP and 293T cells transfected with the β-catenin/TCF responsive pOT construct and mutated pOF control. Cells were also co-transfected with β- catenin. Average relative luciferase activity compared with Renilla for one experiment performed in triplicate is shown. The activity of pOT is greater than pOF in 293T but not LNCaP cells, even with exogenous β-catenin. B – QRT-PCR of β-catenin mRNA expression in LNCaP cells stably transfected with β-catenin shRNA or vector only. There was a significant decrease in average β-catenin levels normalised to 18S (SEM, n=3, t test, ***P<0.001). C – LNCaP stables were cultured in 10% CSS for 72 hrs and then treated with 1 nM R1881 for 24 hrs. QRT- PCR was used to test kallikrein expression. Data represents the average fold change upon R1881 treatment compared with ethanol for each cell line with SEM. A significant reduction in the androgen stimulation of KLK2, KLK3 and KLK4 was observed (n=3, t test, *P<0.05, **P<0.01).

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significantly increased by LiCl compared with KCl treatment and also significantly greater than the pOF negative control vector (Figure 4.2A & B). Of note, LiCl reduced the activity of a KLK3 promoter construct, although comparison with the Basic promoter-less vector control shows that it was only minimally active under these conditions (Figure 4.2C). Since LiCl successfully increased β-catenin:TCF transcriptional activity, the treatments were repeated and changes in endogenous kallikrein mRNA expression were measured using QRT-PCR. Similar to the luciferase assay, KLK3 was down-regulated by LiCl as was the expression of other androgen regulated genes, KLK2, 4 and 15 (Figure 4.2D). However, KLK14 expression was stimulated in both DU145 and 22Rv1 cells implying that it may be a target of β-catenin and TCF.

LiCl can inhibit the activity of other kinases besides GSK3β, albeit with much lower potency

(Davies et al., 2000). Therefore, SB216763, a more specific GSK3β inhibitor with lower IC50 concentration (Coghlan et al., 2000), was used to ensure that changes in KLK14 expression were not due to any off-target effects of LiCl. As expected, SB216763 successfully increased TCF luciferase reporter activity above the DMSO vehicle control in 22Rv1 cells (Figure 4.3A). DU145 and 22RV1 cells were treated with 5 and 20 µM SB216763 for 8 hrs. KLK14 expression was significantly up-regulated at both doses in DU145 cells and with 20 µM SB216763 in 22Rv1 cells cultured in CSS containing media (Figure 4.3B & C). Unlike LiCl treatments, there was no significant change in KLK3 expression (Figure 4.3D). Interestingly, SB216763 treatment also increased KLK14 expression in 22Rv1 cells in the presence of total serum, suggesting that GSK3β inhibition stimulates KLK14 expression regardless of hormone levels (Figure 4.3E). Once again there was no significant change in KLK3 expression (Figure 4.3F).

4.4.3 KLK14 Expression Does Not Correlate With E-cadherin Levels Previous studies have shown that E-cadherin modulates β-catenin signaling in prostate cancer cells (Yang et al., 2002, Verras et al., 2004, Cronauer et al., 2005, Syed et al., 2008). A set of DU145 sublines selected for differences in invasiveness were used to determine whether KLK14 expression correlates with endogenous E-cadherin levels. DU145-E cells are poorly invasive, have rounded morphology typical of epithelial cells and express high levels of E-cadherin. In comparison, the DU145-F subline is highly invasive, has fibroblast-like morphology and low levels of E-cadherin (Chunthapong et al., 2004). The E-cadherin profile of these cell lines was confirmed by Western blot and compared with parental DU145 cells (DU145-P) (Figure 4.4A). Despite the differing E-cadherin levels, there was no change in KLK14 expression between the DU145 sublines (Figure 4.4B left panel). There was also no change in KLK15 expression, the only other prostatic kallikrein expressed in DU145 cells (Figure 4.4B right panel). Thus, no direct correlation between E-cadherin levels and kallikrein expression was observed with this model.

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Figure 4.2. Changes in TCF Activity and Kallikrein Expression in DU145 and 22Rv1 Cells Treated with LiCl. A - DU145 cells were transfected with pOT, pOF, pGL3-basic (BASIC) or KLK3 promoter luciferase constructs and then treated with 40 mM LiCl or KCl control for 24 hrs. Relative luciferase activity compared normalised to Renilla transfection control are graphed with SEM. The pOT promoter construct was significantly stimulated by LiCl compared to KCl. There was also significantly greater activity than the pOF control (One Way Anova, n=3, ***P<0.001). B – 22Rv1 cells were cultured in 2% CSS for 48 Hrs and then transfected and treated in the same manner as DU145 cells. LiCl also significantly increased pOT but not pOF activity in this cell line (One Way Anova, n=6, ***P<0.001). C - PSA promoter activity was significantly reduced in the prescence of LiCl in 22Rv1 cells, whereas empty pGL3 Basic vector was not (t test, n=5, P<0.001). D – QRT-PCR of kallikrein expression in DU145 and 22Rv1 cells treated for 24 hrs with 40 mM LiCl or KCl. Average expression normalised to KCl treatment from one experiment performed with three wells is shown.

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Figure 4.3. Changes in Kallikrein 14 Expression Due to GSK3β Inhibition. A – A luciferase assay of 22Rv1 cells treated with 10 µM SB216763 for 24 Hrs. The graph depicts pOT activity relative to Renilla and normalised to DMSO vehicle control. There was a significant increase in TCF activity in 22Rv1 cells treated with SB216763 (n=3, t test, *P<0.05). B – QRT-PCR of KLK14 expression in DU145 cells treated for 8 hrs with DMSO vehicle control or 5-20 µM SB216763 (n=4, One Way ANOVA with Tukey’s posthoc analysis, **P<0.01). 22Rv1 cells were cultured for 72 hrs in media containing 10% CSS or FCS and then treated for 8 Hrs with SB216763 or DMSO. KLK14 and KLK3 expression was measured with QPCR and plotted in the same manner as DU145 cells. There was a significant increase in KLK14 in both CSS (C) and FCS (E) (P<0.05). There was no significant change in KLK3 expression in either condition (D & F).

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Figure 4.4. KLK14 and KLK15 Expression in DU145 Sublines A – Western blot of E-cadherin levels in DU145-P, -E and –F whole cell lysates. Tubulin was used as a loading control. B – There was no difference in average KLK14 and KLK15 expression in DU145 sublines measured with QRT-PCR in one experiment with three separate wells.

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4.4.4 Induction of KLK14 expression with GSK3β Inhibitors is Independent of β- catenin β-catenin is a prominent target of GSK3β. However, GSK3β is a multifunctional kinase that also phosphorylates other proteins. To determine whether GSK3β inhibition increases KLK14 expression through β-catenin, SB216763 treatment was coupled with β-catenin siRNA knockdown in DU145 cells. In whole cell lysates which contain membrane-bound, cytoplasmic and nuclear protein, β-catenin levels were reduced by β-catenin siRNA compared with the non- targeting control siRNA and mock transfection (Figure 4.5A). Similar reductions were observed in the presence of DMSO and SB216763. The stabilisation of β-catenin levels by SB216763 treatment was more apparent in soluble protein extracts which only contain cytoplasmic and nuclear pools of β-catenin (Figure 4.5B). In these samples, the decrease in β-catenin protein levels with β-catenin siRNA was also more obvious with SB216763 treatment. QRT-PCR results show that RNA interference reduced β-catenin mRNA levels to a similar extent in DMSO and SB216763 treated cells (Figure 4.5C). Therefore, β-catenin levels were successfully attenuated in these experiments. KLK14 expression was next investigated and was once again significantly up-regulated by inhibition of GSK3β. However, this response was not altered in cells depleted of β-catenin (Figure 4.5D). These results demonstrate that although KLK14 expression is regulated by GSK3β, it is unlikely to be a target of β-catenin and TCF.

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Figure 4.5. Kallikrein 14 Expression in DU145 cells with β-catenin Knockdown. DU145 cells were transfected with β-catenin or nontargeting siRNA for 48 hrs and then treated with SB216763 for 8 hrs. Western blots from total cell lysates (A) and soluble fractions (B) depict the decrease in β-catenin protein levels in cells transfected with β-catenin compared with non-targeting control siRNA. C – A significant decrease in β-catenin mRNA levels was measured using QRT-PCR (n=2, One Way ANOVA with Tukey’s posthoc analysis, **P<0.001, ***P<0.001). D - KLK14 expression was also measured with QRT-PCR. It was significantly up-regulated by SB216763 treatment but did not change with β-catenin knockout (*P<0.05). QPCR data is presented as average gene expression normalised to 18S with SEMs.

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4.5 Discussion AR remains essential for the growth and survival of prostate cancer cells even as they become increasingly dedifferentiated and lose other traits of luminal epithelial cells. This means that cross-talk between reactivated developmental signaling pathways and the AR axis is inevitable. The levels of β-catenin, an important signaling molecule in embryonic development and prostate morphogenesis, can be increased by multiple mechanisms in prostate cancer progression (Verras and Sun 2006, Grigoryan et al., 2008, Wang et al., 2008a). The actions of β-catenin depend on the cellular context; it either stimulates the expression of Wnt target genes through TCF, or enhances the transcriptional activity of ligand-bound AR. Importantly, the activity of the TCF and AR axes is mutually exclusive (Cheshire and Isaacs 2003). As prominent androgen- regulated genes, several prostatic kallikreins were likely to be affected by the synergy between β- catenin and AR. Indeed β-catenin was required for maximal KLK2, 3 and 4 expression. Conversely, it was possible that β-catenin and TCF could regulate KLK14 expression since it is repressed by AR. However, KLK14 was up-regulated by GSK3β inhibition but not through β- catenin.

This study demonstrated that depleting β-catenin in LNCaP cells attenuates KLK2, 3 and 4 expression in response to androgens. Although there was no significant difference in KLK15 expression, more subtle changes were expected since it is only modestly androgen regulated. The trend of decreased KLK15 expression in the β-catenin knockout cell line was also observed in cells treated with 10 nM DHT for 4, 8 and 24 hrs (data not shown). Overall, these results demonstrate that the expression of androgen-regulated kallikreins is sensitive to changes in β- catenin levels. Differences in β-catenin signaling might even contribute to the heterogeneity in KLK3 expression that is observed between patients. However, the relative contribution of β- catenin to kallikrein expression in vivo will be difficult establish. One approach would be to use immunohistochemistry on serial tissue sections to determine whether kallikrein staining correlates with β-catenin expression and cellular distribution. Yet there is disagreement between laboratories about whether cytoplasmic and nuclear β-catenin staining increases or decreases in prostate cancer (Gravdal et al., 2007, van Oort et al., 2007, Whitaker et al., 2008). A recent study attributed these discrepancies to technical variation and noted that differences in primary antibodies, antibody concentration and fixation time altered the intensity and specificity of β- catenin staining (Whitaker et al., 2008). Furthermore, cell line experiments suggest that the threshold of β-catenin required to initiate TCF-mediated transcription is below the level of immunodetection (Chesire et al., 2000, Shih et al., 2000). An alternative approach was employed in a study of ovarian endometriod adenocarcinoma specimens that used microarrays to compare the gene expression profiles of tumours with and without Wnt pathway mutations (Schwartz et al., 2003). Notably, an increase of 2.4-fold KLK2 and 2.5-fold KLK3 expression was observed

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in samples with Wnt pathway mutations which presumably had increased β-catenin levels. This implies that β-catenin does indeed stimulate kallikrein expression in vivo. This technique could be used to clarify the role of β-catenin in prostate cancer in future studies.

Unlike the androgen-regulated kallikrein genes, KLK14 expression was not altered by β-catenin even though it was consistently up-regulated by GSK3β inhibition. GSK3β is a multifunctional kinase with several other downstream targets including axin and APC in the β-catenin destruction complex, insulin receptor substrate 1, and the lipogenic enzyme acetyl CoA carboxylase (Doble and Woodgett 2003). The changes in KLK14 expression are probably mediated by the transcription factors that are regulated by GSK3β. Some of these, such as cAMP response element binding protein and micropthalmia-associated transcription factor, are activated by GSK3β so are unlikely to alter KLK14 expression which is stimulated by GSK3β inhibition (Fiol et al., 1994, Takeda et al., 2000, Khaled et al., 2002). Of the transcription factors that are inhibited by GSK3β phosphorylation, c-myc and c-Jun are good candidates for regulating KLK14 (Pulverer et al., 1994, Sears et al., 2000). Both transcription factors have predicted binding sites within the KLK14 proximal promoter that are detected by multiple bioinformatics programs (data not shown). They are also up-regulated during prostate cancer progression (Edwards et al., 2004, Gurel et al., 2008, Ouyang et al., 2008). Notably, regulation of KLK14 expression by c-Jun could underlie its repression by AR. As an activated protein 1 (AP-1) transcription factor, c-Jun homodimerises or heterodimerises with c-Fos. AR inhibits the expression of some c-Jun:c-Fos target genes, possibly by sequestering c-Jun which can act as a coactivator of AR (Shemshedini et al., 1991, Bubulya et al., 1996, Jorgensen and Nilson 2001b, Chen et al., 2006a). Therefore, further studies are warranted to determine whether c-myc and c- Jun regulate KLK14 expression.

Competition between AR and TCF for β-catenin adds an extra layer of complexity to β-catenin signaling in the prostate. Although it was not the focus of this study, some of the data does address this point. LiCl treatment reduced the activity of a KLK3 promoter construct and endogenous KLK2, 3, 4 and 15 expression in 22Rv1 cells cultured in CSS containing media. Under these conditions, AR activity may have been further reduced by TCF sequestering β- catenin. It is also possible that GSK3β inhibition with LiCl altered AR signaling independently of β-catenin because GSK3β directly phosphorylates AR. Whether this stimulates or inhibits AR activity is contentious (Liao et al., 2004, Mazor et al., 2004, Salas et al., 2004, Wang et al., 2004). Down-regulation of prostatic kallikreins with LiCl treatment concurs with studies suggesting that GSK3β enhances AR transcriptional activity. However, this assumes that the decrease in KLK2, 3, 4 and 15 expression was truly independent of β-catenin. Notably, KLK14 was up-regulated by GSK3β inhibition regardless of whether AR was expressed in each cell line.

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Unlike LiCl, there was no statistically significant difference in KLK3 levels with SB216763 treatment, although there was a trend of decreased KLK3 expression when the experiment was conducted in media with total serum. The most likely explanation for the different observations between GSK3β inhibitors is that cells were treated for 24 hrs with LiCl but only 8 hrs with SB216763. The pleotrophic effects of LiCl should also be considered. Although, LiCl is most potent towards GSK3β, it can also inhibit the activity of casein kinase 2 (CK2) and MAPK- activated protein kinase 2 (MAPKAP-K2), which activates p38 MAPK (Davies et al., 2000). Not only do CK2 and p38 both inhibit AR activity, they also suppress β-catenin signaling (Hildesheim et al., 2005, Gioeli et al., 2006, Gotz et al., 2007). These observations emphasise that multiple signaling pathways regulate both AR and β-catenin activity and ultimately determine the extent of cross-talk between these two molecules.

The kallikreins may also alter β-catenin levels. The loss of glandular architecture in prostate cancer leads to the aberrant secretion of kallikreins into the tumour microenvironment where they are exposed to a new range of substrates. One of these is E-cadherin. KLK3, 4, 7 and 14 all cleave a recombinant form of the E-cadherin extracellular domain in vitro (Figure 5.12, Dr Astrid Whitbread, personal communication, Johnson et al., 2007). KLK6 and KLK7 also shed E-cadherin from the membrane in cell culture experiments (Johnson et al., 2007, Klucky et al., 2007). Stable over-expression of KLK3 or KLK4 in PC3 prostate cancer cells causes a loss of E- cadherin, albeit at the mRNA level through an unknown mechanism (Veveris-Lowe et al., 2005). The extracellular domain of E-cadherin is cleaved by several other proteases, including uPA, MMP7, MMP9, ADAM10 and ADAM15 (Plymate et al., 1996a, Plymate et al., 1996b, Davies et al., 2001, Maretzky et al., 2005, Symowicz et al., 2007, Gil et al., 2008, Najy et al., 2008). The 80 kDa soluble E-cadherin fragment promotes the invasion of cancer cells and is detected in the serum of cancer patients (Chunthapong et al., 2004, Maretzky et al., 2005, De Wever et al., 2007, Johnson et al., 2007). However, few studies have investigated whether E- cadherin cleavage alters β-catenin:TCF signaling. One exception is the KLK6 study which found increased levels of nuclear β-catenin in keratinocytes stably-transfected with KLK6 and greater TCF reporter activity in transiently-transfected HEK293 cells (Klucky et al., 2007). Kallikreins could also promote β-catenin signaling via cleavage of IGFBPs. Many kallikreins have been reported to degrade at least one member of the IGFBP family (Cohen et al., 1992, Plymate et al., 1996a, Koistinen et al., 2002, Matsumura et al., 2005, Michael et al., 2006, Borgono et al., 2007b, Sano et al., 2007). This cleavage releases IGF-1 which increases β-catenin stability and co-activation of AR in prostate cancer cells (Verras and Sun 2005). Whether the kallikreins have a significant impact on β-catenin levels in prostate cancer remains to be determined. However, it is possible that there is a positive feedback loop where kallikreins increase β-catenin levels,

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which in turn stimulates kallikrein expression. Once again this would depend on cellular context and the multiple pathways that regulate β-catenin signaling.

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5Chapter 5: Developmental Signalling Pathways in Prostate Cancer: The Expression and Function of the Nodal Axis

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5.1 Introduction Prostate cancer progression is characterised by increasing dedifferentiation. At the tissue level, the degree of dedifferentiation is measured with the Gleason grading system to determine the aggressiveness of prostate cancers and predict patient outcome (Gleason 1977). At the cellular level, tumours acquire a plastic phenotype and mimic developmental processes including epithelial to mesenchymal transition and vasculogenesis (Sharma et al., 2002b, Lawrence et al., 2007). At the molecular level, prostate cancer cells display gene expression signatures of branching morphogenesis and stem cell self-renewal (Glinsky et al., 2005, Schaeffer et al., 2008).

Although prostate cancer recapitulates many aspects of development, it does so in a highly dysregulated manner. Embryonic development, prostate morphogenesis and normal prostate homeostasis are all strictly regulated by bidirectional signals between cells and their microenvironment. These interactions are disrupted in prostate cancer (Stewart et al., 2004). For example, fibroblasts undergo genetic, epigenetic and phenotypic alterations during prostate cancer progression (Tuxhorn et al., 2002, Hill et al., 2005, Hanson et al., 2006) and can in turn induce the malignant transformation of benign prostate epithelial cells (Olumi et al., 1999, Hayward et al., 2001). These changes in cell-cell communication are due to altered expression of paracrine signalling molecules, including factors from the Wnt and TGFβ families, which are deposited in the ECM (Joesting et al., 2005, Li et al., 2008b). Cancer-related proteases, such as kallikreins and MMPs, also modify the tumour microenvironment by remodelling the ECM, releasing growth factors and depositing of pro-migratory cues for cancer cells (Giannelli et al., 1997, Sternlicht et al., 1999, Borgono and Diamandis 2004). Recent studies have modelled the microenvironment using three-dimensional extracellular matrices preconditioned with one cell type and then seeded with another (Seftor et al., 2005, Postovit et al., 2006, Seftor et al., 2006, Postovit et al., 2008b). Using this technique, the inductive cues within a metastatic melanoma conditioned matrix (CMTX) were shown to increase the aggressiveness of non-metastatic melanoma cell lines and cause the transdifferentiation of normal melanocytes to a melanoma-like phenotype (Seftor et al., 2005, Seftor et al., 2006). Similarly, in this study we demonstrate that the plasticity of a poorly invasive DU145 subline, DU145-E, is enhanced when it is cultured on matrices preconditioned with highly invasive DU145-F cells. These observations imply that the phenotype of tumour cells is governed by dynamic interactions with their microenvironment.

Given that the microenvironment can promote tumour progression, the opposite might also be true. Aggressive prostate cancer cells hijack developmental signalling pathways that proceed unchecked because the adult prostate lacks the regulatory mechanisms that strictly control these

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cascades in embryonic development. Therefore, it is possible that restoring the balance of regulatory signals by exposing dedifferentiated prostate cancer cells to a normal embryonic microenvironment may decrease their aggressiveness. To test this hypothesis, DU145 and PC3 cells were exposed to hESC CMTX. Accordingly, the invasiveness and clonogenicity of the prostate cancer cells was attenuated. These results demonstrate that prostate cancer cells are responsive to an embryonic microenvironment and imply that developmental signalling molecules promote the aggressiveness of prostate cancer.

The hESC CMTX model has recently been used to identify the developmental factors acquired by tumour cells. A study with breast cancer and melanoma cell lines identified Lefty A and B, important regulatory molecules in embryogenesis (herein referred to as Lefty), as being responsible for reprogramming tumour cells exposed to hESC CMTX (Postovit et al., 2008b). Lefty exerts these effects by inhibiting Nodal; a TGFβ superfamily member that is not only involved in development but re-expressed by aggressive melanoma and breast cancer cells (Topczewska et al., 2006, Hendrix et al., 2007). Similar to activin members of the TGFβ superfamily, Nodal binds as a dimer to type I (ALK4/ActRIB or Alk7) and type II serine- threonine kinase receptors (ActRIIA or ActRIIB) (Reissmann et al., 2001, Chen et al., 2004). However, Nodal uniquely requires Cripto as a co-receptor to trigger downstream phosphorylation of Smad2 and Smad3 (Shen and Schier 2000). These receptor-regulated Smads then associate with Smad4, translocate to the nucleus, and synergistically activate gene expression with a range of transcription factors (Shi and Massague 2003). In embryos, the strength and localisation of Nodal signalling is tempered by Lefty to ensure correct specification and axial symmetry (Tabibzadeh and Hemmati-Brivanlou 2006). However, Lefty is not expressed in most adult tissues and tumours, allowing Nodal signalling to continue uninhibited (Tabibzadeh et al., 1997, Postovit et al., 2008b). Indeed, Nodal promotes the invasiveness, clonogenicity and tumourigenicity of breast cancer and melanoma cells, effects that are all reversed by hESC CMTX-derived Lefty (Postovit et al., 2008b). Considering that hESC CMTX also reduces the aggressiveness of prostate cancer cell lines, it is possible that Nodal is involved in the progression of prostate cancer. Little is known about the role of Nodal in the prostate. Therefore, to complement the hESC CMTX experiments, the expression and function of the Nodal axis in prostate cancer was also characterised in this study.

Extracellular proteolytic cleavage of the Nodal pro-peptide is necessary for Nodal signalling through the activin receptors. Two serine proteases, furin and PACE4, can activate pro-Nodal in a non-cell autonomous manner in mouse embryos via cleavage of an RXRR motif. Activation of human Nodal in the tumour microenvironment has not been characterised. Given that several

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kallikreins are abundant in the prostate cancer microenvironment and have trypsin-like serine protease specificity, their ability to cleave pro-Nodal was investigated in this project.

In summary, the aim of this chapter is to determine how the microenvironment affects the plasticity of prostate cancer cells using different variations of the CMTX model and investigate the expression, function and activation of Nodal in prostate cancer.

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5.2 Materials and Methods

5.2.1 Cell Culture All prostate cancer cell lines were cultured as previously outlined (Chapter 2.4). COS-1 cells were maintained in RPMI 1640 containing 10% FCS, 50 U/mL Penicillin G, and 50 μg/mL Streptomycin. The aggressive cutaneous melanoma cell line, C8161, was also grown in RPMI 1640 (Welch et al., 1991, Seftor et al., 2002). Poorly aggressive C81-61 cells isolated from the same patient were cultured in Ham’s F10 (Invitrogen) supplemented with 15% FCS, 1x Mito+ (BD Biosciences, North Ryde, NSW, Australia) 50 U/mL Penicillin G, and 50 μg/mL Streptomycin. H9 hESCs were cultured by Dr Lynne-Marie Postovit (Children’s Memorial Research Centre, Chicago) in DMEM-F12 with 20% knockout serum replacer, 1% nonessential amino acids, 1mM L-Glutamine and 4 ng/mL basic fibroblast growth factor (Invitrogen) on a feeder layer of irradiated mouse embryonic fibroblasts (MEFs; strain CF-1; American Type Culture Collection).

5.2.2 Network Formation, DU145 Conditioned Matrices and Periodic Acid-Schiff Staining DU145 CMTX was prepared using a previously described protocol (Seftor et al., 2005, Seftor et al., 2006). Three dimensional matrices were prepared in 12-well plates by adding 25 µL/well of a mixture of rat tail collagen I (average concentration 3 mg/mL; BD Biosciences), 50 µg/mL human collagen IV and 50 µg/mL human laminin (Sigma) which was polymerised with 100% ethanol for 5 min at room temperature. The matrices were washed extensively with 1x PBS before 2 x 105 DU145-P, DU145-F, DU145-E or a 1:1 ratio of DU145-E and DU145-F cells were seeded in complete media. For conditioned matrix experiments, the first set of cells was removed from the matrices after 3 days using 20 mM ammonium hydroxide followed by two washes each of sterile distilled water and PBS. The matrices were examined with light microscopy to confirm they had not detached from the tissue culture plate. The conditioned matrices were then re-seeded with 2 x 105 DU145-E cells for a further 4 days to allow network formation. Polysaccharide and glycoprotein rich vasculogenic networks were visualised using Periodic Acid Schiff’s staining as previously described (Maniotis et al., 1999). Briefly, 3D cultures were fixed with 3.7% formaldehyde for 10 mins, washed twice with distilled water, and then incubated with 0.5% periodic acid solution (Richard Allen Scientific, Kalamazoo, MI, USA) for 6 mins at room temperature. The samples were then rinsed twice with distilled water before Schiff’s reagent (Richard Allen Scientific) was applied for 5 mins at room temperature. Excess stain was removed with repeated washes with distilled water and images where then

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captured using a Hitachi HV-C20 CCD camera (Hitachi Denshi America Ltd., Woodbury, NY, USA) fitted to a Zeiss Televal inverted microscope (Carl Zeiss Inc, Thornwood, NY, USA).

5.2.3 Human Embryonic Stem Cell Conditioned Matrices A previously described technique was used to prepare hESC CMTX with the assistance of Dr Lynne-Marie Postovit and Elisabeth A. Seftor, Children’s Memorial Research Centre, Northwestern University, Chicago (Postovit et al., 2006, Postovit et al., 2008b). Briefly, 200 µL of growth factor-reduced Matrigel (BD Biosciences) was applied to wells of 6-well plates and polymerised for 10 mins at 37 ºC. Colonies of H9 cells were picked with a sterile glass rod, seeded at approximately 5 x 104 cells per well, and cultured in MEF-conditioned media for 3 days. Subsequently, the hESCs were removed with 20 mM ammonium hydroxide followed by washes with warm sterile distilled water, PBS and RPMI 1640. Matrigel only control matrices, which were not conditioned with hESCs, were also treated with ammonium hydroxide and washed with water. The matrices were always examined with light microscopy to confirm that they were still attached to the tissue culture plates. The denuded matrices were re-seeded with 2.5 x 105 DU145 or PC3 cells for a further 3 days. For comparison, cells were also seeded onto Matrigel alone which had been processed the same as hESC CMTX. For functional assays, the prostate cancer cells were harvested with trypsin whereas they were collected together with the hESC CMTX for protein analyses using M-PER lysis buffer (Pierce). The network formation of cells collected from hESC CMTX was assessed by seeding 5 x 106 cells onto fresh Matrigel for 4 days.

5.2.4 Soft Agar Assays Clonogenicity was assessed in 6-well plates coated with RPMI 1640 containing 10% FCS and 0.5% agar. Stably transfected LNCaP cells or DU145 cells harvested from conditioned matrices were seeded at 5 x 103 cells per well in media containing 0.35% agarose, grown until macroscopic, stained with 0.001% crystal violet, and the total number of colonies in each well counted.

5.2.5 Zymography To compare pro-MMP2 expression, whole cell lysates spiked with non-reducing sample buffer (final concentration of 25 mM Tris pH 6.8, 5% glycerol, 0.25% SDS and 0.0.5% bromophenol blue (w/v)) were separated on 8% polyacrylamide gels containing 1 mg/mL porcine gelatin (Sigma) in 375 mM Tris pH 8.8. After electrophoresis, gels were washed twice for 15 mins with 2.5% Triton X-100, rinsed 5 times with distilled water, and then incubated overnight at 37 ºC in

5 mM CaCl2, 50 mM Tris pH 7.0 to activate the enzymes. To visualise gelatine digestion, the

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gels were stained with 0.4% Coomassie blue, 40% methanol and 10% acetic acid and then destained with 30% methanol and 10% acetic acid.

5.2.6 RT-PCR and QRT-PCR Total RNA extractions and cDNA synthesis were conducted according to standard procedures as was RT-PCR amplification of Lefty A/B, HPRT1, Pitx2, GAPDH, furin and PACE4 (Chapters 2.5 & 2.6). Primers sequences are listed in Appendix C. Note that Lefty primers anneal to both Lefty A and Lefty B which have 96% sequence identity (Tabibzadeh and Hemmati-Brivanlou 2006). The Lefty A/B experiment was performed by Dr John Lai (QUT).

5.2.7 Immunohistochemistry Formalin-fixed paraffin-embedded prostate specimens were obtained from the Pathology Core Facility of the Northwestern University Prostate Cancer Spore and the Australian Prostate Cancer Collaboration BioResource. The histopathology of specimens was examined by three urological pathologists, Drs Angus Collins, Kris Kerr and Megan Turner (Sullivan and Nicolaides Pathology, Brisbane) who assigned Gleason scores by consensus. Immunohistochemistry was performed by Dr Naira V. Margaryan, Children’s Memorial Research Centre, Northwestern University, Chicago according to a protocol previously used to detect Nodal in breast cancer and melanoma specimens (Topczewska et al., 2006, Postovit et al., 2008b). Specimens were dewaxed with xylene (2 x 2 mins), rehydrated with a graded series of ethanol (100% for 2 x 1 min, 90% for 1 min, 70% for 1 min and 50% for 1 min), and rinsed with TBS-T for 5 mins. A decloaking chamber (Biocare Medical, Walnut Creek, CA) and citrate buffer pH 6.0 (Lab Vision, DKSH Australia, Hallam, Vic, Australia) was used for antigen retrieval. Subsequent steps were performed on a HNS 710i Automated Immunostainer (Richard- Allan Scientific). Four blocking solutions, 0.03% 0.03% hydrogen peroxide (RAS), avidin and biotin block (Avidin/Biotin Blocking Kit, Vector Laboratories, Inc., Burlingame, CA, USA), and a serum-free protein block (Richard-Allan Scientific) were each applied for 10 mins. Samples were incubated with anti-Nodal primary antibody (20 µg/mL; R&D Systems) or ChromPure normal goat IgG (Jackson ImmunoResearch Laboratories, West Grove, PA, USA) for 90 mins and then washed with TBS-T. Biotinylated anti-goat secondary antibodies and a peroxidase- conjugated streptavidin tertiary (Richard-Allan Scientific) were each applied for 20 mins. Specimens were then incubated with DAB as the chromogen and counterstained with Mayer’s Haematoxylin (Richard-Allan Scientific).

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5.2.8 Recombinant Nodal Treatment DU145 and LNCaP cells were grown to 50% confluence in 6-well plates and then cultured overnight in serum-free RPMI 1640 media. Cells were treated with 10 to 1000 ng/mL recombinant mature Nodal (R&D Systems) or vehicle control (4 mM HCl, 0.1% BSA) for 60 mins unless otherwise noted. TGFβ1 (1 ng/mL; R&D Systems) was used as a positive control for Smad2 phosphorylation. In some experiments, cells were also treated with 10 µM SB-431542, an Alk4/5/7 inhibitor, or DMSO vehicle control. This concentration of SB-431542 has previously been shown to inhibit Alk4/5/7 signalling in human cell lines (Inman et al., 2002). Protein lysates were collected using the previously described procedure (Chapter 2.10) except that 2 mM NaF and 10 mM Na3VO4 were added to the lysis buffer to inhibit phosphatase activity.

5.2.9 Stable Transfections Human pre-pro-Nodal was amplified from H9 hESC cDNA (Fwd: 5’ TCCCTCCAGGATGTCTCGAGAGGCACCCAC 3’, Rev: 5’ TTCAGGATCCGCCAGCC CACCATGCACGCC 3’) and cloned into the pcDNA3.1 Flag-His vector using BamHI and XhoI restriction endonuclease sites (Invitrogen). Inserts were sequenced at the Australian Genome Research Facility, Brisbane, Australia. The epitope tags were at the C-terminus of Nodal. T25 flasks of early passage LNCaP and PC3 cells were transfected with 10 µg of Nodal or vector only plasmid DNA using Lipofectamine 2000 (Invitrogen) according to the standard protocol (2.12). After 48 hrs, the media was supplemented with 800 µg/mL geneticin (Invitrogen) to select for stable transfectants which took approximately 10 days. The stably cell lines were then maintained in media containing 400 µg/mL geneticin.

5.2.10 Luciferase Assays and R1881 Treatment LNCaP cells were seeded at a density of 6 x 104 cells per well in 24-well plates and cultured in phenol red-free RPMI 1640 medium containing 10% charcoal-stripped serum for 72 hrs. Each well was transfected with 300 ng of the KLK3 luciferase promoter construct (Lai et al., 2007), 300 ng of Renilla and 10-250 ng of Nodal pcDNA3.1Flag-His with 2 µL of Lipofectamine 2000 using the previously described protocol (Chapter 2.12). Empty pcDNA3.1 Flag-His vector was also added to ensure equimolar amounts of total plasmid DNA were transfected between treatments. After 6 hrs of transfection, cells were treated with 1 nM R1881 or ethanol vehicle control for 24 hrs. Luciferase activity was quantified using the Dual-Luciferase Reporter Assay System on a PolarStar plate reader.

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5.2.11 Nodal and Kallikrein Co-transfections COS-1 cells were grown to 90% confluency in 6-well plates. Pre-pro-KLK2, 3, 4, 6 and 14 constructs in the pcDNA3.1 vector were provided by Dr Tara Veveris-Lowe, Dr Tracey Harvey, Dr Rachael Collard, Ms Janet Reid and Ms Satomi Okano, QUT. Proteolytically inactive mutant forms of each kallikrein with the catalytic serine substituted for an alanine residue were used as negative controls. To account for differences in the size of inserts, each well was transfected with equal copies of plasmid DNA; 1 pM of pcDNA3.1 only, 0.75 pM of Nodal pcDNA3.1 Flag-His plus 0.25 pM of empty vector, or 0.75 pM of Nodal with 0.25 pM of each kallikrein construct. For each well, plasmid DNA and 8 µL of Lipofectamine 2000 were diluted to a total volume of 500 µL in Optimem in accordance with the standard transfection protocol (Chapter 2.12). The transfection mix was added to 1 mL of serum and antibiotic free RPMI 1640 and the cells were cultured for an additional 48 hrs. Whole cell lysates were extracted as previously described (Chapter 2.10). Conditioned media was collected and centrifuged to remove cellular debris and the protein was precipitated overnight at -20 ºC with 9 volumes of 100% ethanol. Samples were then centrifuged at 4g for 10 mins at 4ºC to collect the precipitate which was resuspended in lysis buffer.

5.2.12 Recombinant Kallikrein Activation Recombinant human pro-KLK4 produced in SF9 insect cells was purified by Mr Carson Stephens, QUT, while pro-KLK14 was purchased from R&D Systems. For enzyme activation, pro-KLK4 and pro-KLK14 were incubated at 100 ng/µL at an 80:1 molar ratio with thermolysin (1.8 ng/uL; Sigma) in buffer containing 50 mM Tris, 150 mM NaCl and 0.05% Brij-35 (Sigma). The reaction proceeded for 1 hr at 37 ºC before thermolysin activity was inhibited with 200 µM phosphoramidon (Sigma). KLK3 purified from seminal plasma (Sigma) was already partially enzymatically active (personal communication Dr Scott Stansfield). Pre-activated KLK4, depleted of thermolysin by liquid chromatography was provided by Mr Carson Stephens and Dr Scott Stansfield, QUT. The specific concentration of active KLK4 in these samples was measured using a fluorometric peptide substrate and a standard curve of the serine protease inhibitor aprotinin.

5.2.13 E-Cadherin:Fc and Fibronectin Digests Recombinant E-cadherin ectodomain fused to the Fc region of human IgG was purchased from R&D Systems. E-cadherin:Fc (100 ng) was digested with 30 ng of activated KLK4 or KLK14 in 100 mM Tris pH 7.5, 100 mM NaCl, 2.5 mM EDTA, 0.02% Tween-20 and 200 µM phosphoramidon for 20 mins and 2 hrs at 37 ºC. The same protocol was used for KLK3 except that the reactions were incubated for 2 hrs and overnight. The approximate molar ratio of

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kallikrein to E-cadherin:Fc was 1:20. E-cadherin:Fc digestion was monitored using Western blots. Fibronectin (500 µg per reaction) was incubated with 25 ng of KLK4 or KLK14 for 30 mins and 2 hrs at 37 ºC in the same buffer as E-cadherin:Fc. The molar ratio of kallikrein to fibronectin was approximately 1:300. KLK3 reactions were carried out with 50 ng of enzyme (approximate molar ratio of 1:150) for 2 hrs and overnight. Fibronectin digests were separated on 7% polyacrylamide gels and visualised with silver staining. For both substrates control reactions were performed where kallikreins were omitted or replaced by the thermolysin and phosphoramidon activation mix.

5.2.14 Silver Staining Silver staining was performed using a previously reported methodology (Heukeshoven and Dernick 1985). Polyacrylamide gels of fibronectin digests were fixed in 40% ethanol and 10% acetic acid in distilled water for 30 mins. The gels were then incubated for 30 mins in sensitising solution (30% ethanol, 6.8% sodium acetate (w/v) and 0.5% sodium thiosulphate (w/v)), washed with distilled water three times for 10 mins and then incubated with silver solution (0.25% silver nitrate (w/v) and 0.015% formaldehyde) for 20 mins. After two 1 min washes with distilled water, the gels were incubated with developing solution (2.5% sodium carbonate (w/v) and 0.0074% formaldehyde) for 5 min to visualise silver stained proteins.

5.2.15 Nodal Purification Pro-Nodal was purified from the conditioned media of COS-1 cells stably or transiently transfected with pcDNA3.1 Nodal Flag-His. Stable cell lines were generated using the same methodology as LNCaP and PC3 cells. In addition, T175 flasks of confluent COS-1 cells were transiently transfected for 48 hrs with 17.5 µg of Nodal using 50 µL of Lipofectamine 2000 in a total volume of 5 mL OptiMem. For each batch of purifications, conditioned media, either serum-free or containing 5% serum, was collected from 8 to 16 T175 flasks. The media was centrifuged at 2000 x g to pellet any cellular debris and then dialysed overnight at 4 ºC in NPI buffer (30.8 mM NaH2PO4, 47.3 mM Na2HPO4 and 300 mM NaCl, pH 8.0) containing 10 mM imidazole (NPI-10). After dialysis, 1 µL of Ni-NTA resin (Qiagen) washed with NPI-10 buffer was added per millilitre of conditioned media. The solution was incubated for 4 hrs at 4 ºC on a rotary mixer and then passed twice through a column (Biorad). The resin bed was then washed twice with NPI-10 buffer at 10 times the resin volume (approximately 2-4 mL). After 3 washes of the same volume with NPI buffer containing 20 mM imidazole (NPI-20), pro-Nodal was eluted with NPI buffer containing 250 mM imidazole in 500 µL fractions. The first 2 or 3 elutions were pooled and concentrated using 10 kDa cut-off filter columns (Millipore) centrifuged at 14 000 x g at 4 ºC. The samples were then buffer exchanged by passing three PBS

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washes through the filter devices. The concentration of pro-Nodal was calculated using Western blots with a standard curve of recombinant mature Nodal. Total protein concentration was quantified using the bicinchoninic assay (Chapter 2.10) which was modified for small volumes by adding 2 µL of working reagent to 1 µL of sample. Absorbances were measured using a NanoDrop 1000 spectrophotometer.

For some experiments pro-Nodal was further purified by immunoprecipitation using a NHS HP SpinTrap kit (GE Healthcare) according to the manufacturer’s protocol. Coupling buffer (0.15 M

triethyl ammonium hydrogen carbonate (NaHCO3), 500 mM NaCl, pH 8.3) was used to immobilise 8 µg of polyclonal rabbit anti-human Nodal antibody to the NHS-activated sepharose. Residual active groups were then blocked with alternative washes of two buffers; 0.5 M ethanolamine, 0.5 M NaCl, pH8.3 and 0.1 M acetate, 0.5 M NaCl, pH 4.0. Pro-Nodal was then incubated with the resin overnight on a rotary mixer at 4 ºC. The columns were washed three times with TBS containing 2 M urea, pH 7.5 and then pro-Nodal was eluted with 0.1 M acetic acid. The eluant was then reconcentrated and buffer exchanged as before.

5.2.16 Pro-Nodal Digestions Ni-NTA purified pro-Nodal (4 ng per reaction) was incubated overnight at 37 ºC with 2 ng of KLK4 and KLK14 or 8 ng of KLK3 in the same reaction buffer as E-cadherin:Fc and fibronectin reactions. The molar ratio of KLK4 and KLK14 to pro-Nodal was 1:4.8, and 1:1.2 for KLK3. In subsequent experiments, 9 ng of Ni-NTA purified or immunoprecipitated pro-Nodal was digested with active KLK4 in molar ratios of 1:1 to 1:1000 (pro-Nodal:KLK4) overnight at 37 ºC. Nodal digests were analysed with Western blots using an anti-Flag primary antibody.

5.2.17 Western blots All cell lysates were collected as previously outlined (Chapter 2.10) unless otherwise noted. Western blots for all experiments were conducted as previously described (Chapter 2.11) except for samples from recombinant kallikrein and pro-Nodal enzyme:substrate assays. These samples were separated with precast 12% Bis-Tris polyacrylamide gels (Invitrogen) using 1x NuPAGE MES SDS running buffer (Invitrogen) for 30 mins at 200 V. The protein was transferred onto nitrocellulose membranes with buffer containing 25 mM Bis-Tris, 25 mM tricine, 1.025 mM EDTA and 12% methanol and then probed with antibodies as previously described (Chapter 2.11). All primary antibodies are listed in Appendix D and were diluted in Odyssey buffer, detected with donkey-anti-mouse-800 or goat-anti-rabbit-680 fluorescent secondary antibodies and visualised using a LI-COR Odyssey scanner. Laminin 5 γ2 and actin are two exceptions which were diluted in 5% skim milk powder in TBS-T. These blots were then probed with

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horseradish peroxidise-conjugated goat-anti mouse or rabbit secondary antibodies and developed using SuperSignal West Pico Chemiluminescent Substrate (Pierce) with X-Ray film.

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5.3 Results

5.3.1 The Microenvironment Regulates the Plasticity of Prostate Cancer Cells Interactions between prostate cells and the microenvironment were investigated using the previously described CMTX technique. One cell line was seeded onto a matrix of collagen I, collagen IV and laminin for 72 hrs, removed with ammonium hydroxide, and then replaced with another. Poorly invasive (DU145-E), highly invasive (DU145-F) and parental (DU145-P) DU145 cells were used. The ability of cells to undergo vasculogenic mimicry was used to measure changes in cellular plasticity. Vasculogenic mimicry involves the formation of fluid conducting networks by tumour cells and is analogous to the development of primitive channels during vasculogenesis in embryos (Maniotis et al., 1999, Hendrix et al., 2003). As shown in Figure 5.1A, parental DU145 cells (DU145-P) are able to form back-to-back loops typical of vasculogenic mimicry. Periodic acid-Schiff staining was used to highlight these ECM-rich networks. DU145-E and DU145-F cells did not form networks separately but were able to when they were co-cultured in a 1:1 ratio (Figure 5.1B-D). Therefore, vasculogenic mimicry requires dynamic interactions between different subpopulations of DU145 cells. Significantly, DU145-E cells exposed to DU145-F CMTX were able to form back-to-back loops similar to parental cells (Figure 5.1E). As a control, DU145-E cells were cultured on conditioned matrices from other flasks of DU145-E cells (Figure 5.1F). They did not undergo vasculogenic mimicry under these conditions. The denuded DU145-E and DU145-F matrices were both free of cells (Figure 5.1E & F insets). These experiments indicate that the plasticity of prostate cancer cells is modulated by the microenvironment and establish the CMTX technique as a useful model for investigating heterotypic interactions between different populations of cells.

5.3.2 Human Embryonic Stem Cell Conditioned Matrices Reduce the Aggressiveness of Prostate Cancer Cells The CMTX model was next used to investigate whether aggressive prostate cancer cells harnessing developmental signalling pathways would respond to regulatory cues from hESCs. DU145 and PC3 cells were used for these analyses. Both cell lines are highly tumourigenic, have an interconverted phenotype with vimentin expression and contain a subpopulation of stem-like cells that form holoclones (Sobel and Sadar 2005b, Sobel and Sadar 2005a, Wei et al., 2007, Li et al., 2008a). The hESC CMTX was prepared with Matrigel which supports the growth of hESCs better than the defined matrix of collagen I, collagen IV and laminin (Figure 5.2A). Similar to previous experiments, the hESCs were cultured on Matrigel for 72 hrs before being removed with ammonium hydroxide. The denuded CMTX is shown in Figure 5.2A (inset). DU145 and PC3 cells were then exposed to hESC CMTX or Matrigel alone for 72 hrs.

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Figure 5.1. Vaculogenic Mimicry of DU145 Sublines A - DU145 cells formed vasculogenic networks when grown on collagen I/collagen IV/laminin matrices. PAS staining was used to highlight characteristic back to back loops (noted with arrows). B –DU145-E to DU145-F cells also formed networks when cultured in a 1:1 ratio. C – DU145-E cells alone do not exhibit vasculogenic mimicry. D – DU145-F cells also fail to form back to back loops alone. E – DU145-E cells undergo vasculogenic mimicry when cultured on DU145-F CMTX. F – As a control, DU145-E cells were also cultured on their own CMTX. There was no network formation in this situation. All cells were removed from DU145-F and DU145-E CMTX preparations (E & F insets). Scale bars equal 20 µm.

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Figure 5.2. Human Embryonic Stem Cell-Conditioned Matrices Decrease the Aggressiveness of DU145 and PC-3 Cells A – hESCs form dense masses with small cell bodies on Matrigel. The hESC CMTX was free of cells (A – inset). B – DU145 cells harvested from Matrigel and replated onto fresh Matrigel for 3 days form back to back loops that are characteristic of vasculogenic mimicry. C – Vasculogenic mimicry is abrogated in DU145 cells that are first cultured on hESC CMTX. D – Zymography of pro-MMP2 and Western blots of laminin 5 γ2 (L5 g2) chain reveal decreased expression in DU145 and PC-3 cells cultured on hESC CMTX. Actin was used as a loading control. E – Average colony formation in soft agar assays normalised to cells cultured on Matrigel. Representative data from one of two experiments with 6 replicates each is shown. Scale bar equals 100 μm.

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Differences in vasculogenic mimicry were assessed by harvesting the cells and re-seeding them onto fresh Matrigel for 48 hrs. As shown in Figure 5.2B & C, compared with cells cultured on Matrigel alone, the ability of DU145 cells from hESC CMTX to form back-to-back loops was attenuated. The networks were discontinuous and poorly formed. The levels of MMP2 and laminin 5 γ2 chain, two factors required for vasculogenic mimicry, were also examined (Seftor et al., 2001). Zymograms revealed decreased pro-MMP2 expression and Western blots showed reduced laminin 5 γ2 chain levels in extracts of DU145 and PC3 cells exposed to hESC CMTX (Figure 5.2D). The clonogenicity of cells harvested from conditioned matrices was also compared using soft agar assays. There was a marked reduction in the anchorage-independent growth of cells exposed to hESC CMTX; 91% for DU145 cells and 75% for PC3 cells (Figure 5.2E). The in vitro invasiveness of DU145 cells decreased by 34% when they were exposed to hESC CMTX, whereas there was only a slight 9% reduction for PC3 cells (personal communication, Elisabeth Seftor, Children’s Memorial Research Centre, Chicago). Nevertheless, the changes observed in vasculogenic mimicry, clonogencity and invasiveness suggest that the embryonic microenvironment can reduce the plasticity and aggressiveness of prostate cancer cells.

5.3.3 Human Embryonic Stem Cell Conditioned Matrices Decrease Nodal Expression Previous studies have shown that hESC CMTX also attenuates the vascologenic mimicry and clonogenicity of breast cancer and melanoma cell lines (Postovit et al., 2006, Postovit et al., 2008b). Further analyses revealed that this effect was due to Lefty, a highly abundant protein in hESC CMTX that potently inhibits Nodal, an embryonic morphogen re-expressed by aggressive tumour cells (Postovit et al., 2008b). One of the actions of Lefty in embryos and tumour cells exposed to hESC CMTX is that it breaks the auto-induction loop of Nodal signalling that stimulates Nodal expression. Therefore, Western blots were used to determine whether changes in Nodal expression contribute to the response of prostate cancer cells to hESC CMTX. As shown in Figure 5.3, there was a 44% decrease in Nodal protein in DU145 cells cultured on hESC CMTX compared with Matrigel alone. Although Nodal was also expressed by PC3 cells, there was no change in Nodal levels in cells from hESC CMTX. This data shows that decreased Nodal levels correlate with the reduced plasticity of DU145, but not that of PC3 cells, and is the first observation of Nodal expression in prostate cancer.

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Figure 5.3. Decreased Nodal Expression in DU145 Cells Cultured on hESC CMTX A – Western blot of Nodal in DU145 and PC3 cells cultured on Matrigel or hESC CMTX for 72 Hrs. Densitometry was used to compare Nodal levels with GAPDH and tubulin loading controls. There was an average 44% decrease in Nodal expression in DU145 cells cultured on hESC CMTX versus Matrigel alone. There was no reduction in PC3 cells.

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5.3.4 The Nodal Axis is Expressed in Prostate Cancer Cells Nodal is expressed by breast, colon and testicular cancer and melanoma cell lines and regulates invasiveness, clonogenicity and in vivo tumour formation (Adkins et al., 2003, Topczewska et al., 2006, Postovit et al., 2008a). Therefore, the expression and function of Nodal in prostate cancer warranted further investigation. The specificity of Nodal Western blots was first verified. Nodal is highly expressed in hESCs (Besser 2004, Vallier et al., 2005, Smith et al., 2008) and was predominantly detected as a 35 kDa band representing pro-Nodal using a rabbit anti-human Nodal primary antibody (Figure 5.4A). This size is consistent with previous reports (Constam and Robertson 1999, Beck et al., 2002, Blanchet et al., 2008). The 50 kDa band present in hESCs may be pre-pro-Nodal and the 25 kDa band mature Nodal. The molecular weights of these bands are also similar to other studies. Other cell lines were probed with both the rabbit anti-human Nodal antibody and a goat-anti mouse Nodal antibody (Figure 5.4A). Human and mouse Nodal share 80% sequence identity within the mature region to which the antibodies were raised (Siegelman and Weissman 1989). Melanoma cell lines, C8161 and C81-61, were used as additional controls. Like previous reports, metastatic C8161 cells expressed high levels of Nodal compared with C81-61 cells (Topczewska et al., 2006, Postovit et al., 2008a). The 35 kDa form of Nodal was detected with both antibodies. The expression Flag-His-tagged Nodal transfected into COS-1 cells was also examined. Transfected Nodal was detected by both antibodies and, allowing for differences due to the epitope tags, was a similar size to the endogenous protein. Intriguingly, as has been observed in previous studies, Nodal was detected as both a single band and a doublet (Topczewska et al., 2006, Postovit et al., 2008b). This varied within experiments for different samples and between experiments for the same sample and is possibly due to differences in glycosylation (Blanchet et al., 2008). Nodal expression in LNCaP, PC3 and DU145 prostate cancer cell lines was also tested alongside the controls. Similar to the hESC CMTX Western blot, Nodal was detected in PC3 and DU145 samples. The 35 kDa band was not observed in LNCaP cells. These experiments demonstrate that pro-Nodal can be specifically detected in prostate cancer cells.

The expression of key factors in the Nodal axis was next examined using a broader panel of prostate cell lines. Similar to previous Western blots Nodal was detected in PC3 and DU145 lysates (Figure 5.4B). Notably, Nodal was not expressed in any of the other prostate cell lines. Faint bands were sometimes observed in LNCaP, 22Rv1 and MDA-PCa-2b samples, however the molecular weight of these bands differed from the positive controls. In contrast Cripto, which acts as a co-receptor for Nodal signalling through ActRIIA/B and Alk4/7, was expressed in all prostate cell lines. The 38 kDa Cripto band is of a similar size to other studies

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Figure 5.4. Expression of the Nodal Axis in Prostate Cell Lines A – Western blots with rabbit anti-human Nodal and goat anti-mouse Nodal primary antibodies. Endogenous pro-Nodal was detected as a 35 kDa band with both antibodies in H9 hESC, PC3, DU145 and C8161 lysates. A small amount was also observed in C81-61 samples. Nodal was detected as a 40-42 kDa doublet in lysates of COS-1 cells transiently transfected with Nodal FlagHis. B – Western blot of prostate cell lines for Nodal, Cripto and GAPDH loading control. Pro-Nodal was only detected in PC-3 and DU145 cells with the anti-human Nodal antibody. The 38 kDa Cripto band was observed in all cell lines. Representative data from 2 experiments is shown with the GAPDH panel corresponding to the Cripto blot. C – RT-PCR for LeftyA/B and HPRT1 house-keeping gene. Lefty was only amplified from H9 hESC cDNA. NTC = no template control.

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(Brandt et al., 1994, Ciardiello et al., 1994, Xing et al., 2004, Postovit et al., 2008b) and was also present in H9 hESC lysates and DU145 membrane preparations (data not shown).

This result is also consistent with a previous report that DU145 and LNCaP cells express the same amount of Cripto protein (Xing et al., 2004). Using RT-PCR, Lefty expression was also analysed. Unlike Nodal and Cripto, no prostate cell lines expressed Lefty except for a faint signal in MDA-PCa-2b cells when the reaction was extended to greater than 40 cycles (Figure 5.4C). H9 hESC cDNA was used as a positive control and the specificity of the band was confirmed with DNA sequencing.

5.3.5 Nodal Expression in Prostate Tissue Given that Nodal was detected in prostate cancer cell lines, immunohistochemistry was used to examine Nodal expression in prostate tissue. There was little Nodal immunoreactivity in benign samples compared with the intense cytoplasmic staining of epithelial cells in Gleason grade 3+3, 4+4, and 4+5 prostate cancer (Figure 5.5). There was negligible immunoreactivity in serial sections probed with the isotype control antibody. Specimens with adjacent benign and malignant glands were examined to confirm that changes in Nodal expression were not simply due to heterogenous staining between patients. As shown in Figure 5.6, Nodal expression was consistently higher in areas of Gleason grade 3 and 4 prostate cancer compared with benign glands.

5.3.6 Prostate Cancer Cells are Responsive to Nodal Prostate cancer cells were treated with recombinant mature Nodal (rNodal) to confirm that the Nodal pathway is also functionally active. DU145 cells which express Nodal and Cripto and LNCaP cells which only express Cripto were used for these experiments. Nodal binding to the Cripto, ActIIRA/B and Alk4/7 complex triggers Smad2 phosphorylation in mammalian embryos, hESCs and cancer cell lines (Kumar et al., 2001, Yeo and Whitman 2001, Topczewska et al., 2006). Nodal also induced Smad2 phosphorylation in DU145 cells (Figure 5.7A). Densitometry was used to normalise pSmad2 to total Smad2 levels. In time course experiments with 1 µg/mL rNodal, increased pSmad2 levels were detected after 30 mins with maximal response after 60 mins (Figure 5.7A). Recombinant TGFβ1 was used as a positive control and was more potent than Nodal, stimulating greater Smad2 phosphorylation at a concentration of 1 ng/mL. Therefore, dose response experiments were used to test lower concentrations of rNodal. Only a small increase in pSmad2 was observed with 10 and 100 ng/mL rNodal whereas 500 and 1000 ng/mL resulted in marked phosphorylation (Figure 5.7B). Although relatively high concentrations of rNodal were needed to trigger Smad2 phosphorylation, this response was

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Figure 5.5. Nodal Expression in Prostate Tissue Immunohistochemistry was used to examine Nodal expression in human prostate tissue specimens. Two representative cases of benign prostate and Gleason score 3+3, 4+4 and 4+5 prostate cancer are shown. There is more intense staining in malignant glands. Portions of serial sections probed with the goat isotype control antibody are shown in panels to the right.

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Figure 5.6. Expression of Nodal in Adjacent Regions of Benign and Malignant Prostate. Six representative samples of prostate tissue with benign glands adjacent to Gleason grade 3 or 4 prostate cancer stained for Nodal using immunohistochemistry. There is more intense staining in malignant glands.

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Figure 5.7. Nodal Stimulates Smad2 Phosphorylation in Prostate Cancer Cells A – DU145 cells were treated for 0-60 mins with 1000 ng/mL recombinant Nodal or 30 min with 1 ng/mL TGFβ1. Western blots were used to compare pSmad2 and total Smad2 levels. Relative pSmad2 versus total Smad2 levels were measured using densitometry. The graph represents average values from 2 experiments with SEM. Nodal significantly increased Smad2 phosphorylation at 60 mins (One Way Anova with Tukey’s posthoc analysis, n=2, *P<0.05). B – DU145 cells were treated for 60 min with 0-1000 ng/mL Nodal and 10 µM SB431542 (+) or DMSO control (-). LNCaP cells were treated with 500 ng/mL Nodal for 6 hrs with fresh Nodal added every 2 Hrs. The graphs depict average pSmad2 versus total Smad2 levels from 3 experiments. There was a significant increase in pSmad2 with 1000 ng/mL Nodal in DU145 cells and significant decrease when SB431542 was added (One Way Anova with Tukey’s posthoc analysis, n=3, ***P<0.001). There was also a significant increase in Smad2 phosphorylation in LNCaP cells with Nodal treatment (t test, n=3, **P<0.01).

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specific because the Alk4/5/7 inhibitor SB-431542 abrogated Nodal-induced Smad2 phosphorylation (Figure 5.7B). This confirms that Smad2 phosphorylation was not due to low affinity binding of Nodal to non-physiological receptors. LNCaP cells exhibited an even more modest response to rNodal with 1 hr treatment compared with DU145 cells (data not shown). Yet a significant increase in pSmad2 was observed when cells were treated with 500 ng/mL rNodal for 6 hrs with fresh ligand added every 2 hrs (Figure 5.7B). Therefore, prostate cancer cells are responsive to Nodal, although Nodal signalling is less intense compared with other TGFβ ligands.

5.3.7 Nodal Increases the Clonogenicity of Prostate Cancer Cells Decreased Nodal expression correlated with reduced plasticity of DU145 cells cultured on hESC CMTX. To complement these experiments, the effect of increased Nodal expression was also investigated. LNCaP and PC3 cells were transfected with a construct encoding pre-pro-Nodal with Flag and His tags or pcDNA3.1 vector control. After antibiotic selection, the expression of Nodal in the polyclonal stable transfectants was confirmed with Western blots (Figure 5.8A). Soft agar assays were then used to compare the clonogenicity of these cell lines. There was a significant increase in the number of colonies for Nodal-transfected LNCaP cells compared with the vector control (Figure 5.8B). The increase in anchorage-independent growth was also apparent at the macroscopic level (Figure 5.8B, lower panel). There was no significant difference in the clonogenicity of PC3 cells which also express endogenous Nodal. In keeping with the low potency of Nodal signalling, there was minimal baseline Smad2 phosphorylation in the Nodal expressing LNCaP cells (data not shown). Nevertheless, the de novo expression of Pitx2, a well known Nodal target gene in the left lateral plate mesoderm of embryos (Yoshioka et al., 1998, Essner et al., 2000, Shiratori et al., 2001), confirmed that the Nodal axis was functionally active (Figure 5.8C). Two other downstream genes from embryonic mesoderm were also tested: brachyury, which requires low levels of Nodal signalling and goosecoid, which is up-regulated by high levels of Nodal (Gritsman et al., 2000). Regardless of Nodal transfection, neither gene was expressed in LNCaP cells although they were successfully detected in H9 hESCs which were used as a positive control (data not shown).

5.3.8 Nodal Antagonises Androgen Receptor Signalling As noted for β-catenin, developmental signalling molecules can modulate the activity of the AR axis in prostate cancer cells. Indeed, there are several layers of cross-talk between AR and the TGFβ superfamily (Danielpour 2005). To investigate whether Nodal influences AR signalling, LNCaP cells were co-transfected with a KLK3 promoter construct and pre-pro-Nodal or vector

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Figure 5.8. Nodal Increases LNCaP Clonogenicity A – Western blot with anti-Flag primary and GAPDH primary antibodies confirming Nodal expression in stably transfected LNCaP and PC-3 cells. B – Relative clonogenicity of Nodal and vector only cell lines in soft agar assays. The graph depicts average data with SEM from 3 experiments normalised to vector only cells. There was a significant increase in the clonogenicity of LNCaP cells stably transfected with Nodal compared to the vector control (t test, n=3, P<0.05). Phase contrast images of crystal violet stained cells confirm the increased number of colonies in Nodal transfected LNCaP cells compared with vector control. In contrast there was similar numbers of colonies in the PC-3 cell lines. C – RT-PCR revealing de novo Pitx2 expression in LNCaP cells stably transfected with Nodal. NTC = no template control.

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Figure 5.9. Nodal Antagonises Androgen Receptor Activity A – Luciferase assay with KLK3 pGL3 vector and 0, 10, 50 and 250 ng of Nodal pcDNA3.1 per well. Cells were cultured in 10% CSS for 72 hrs, transfected for 8 hrs and then treated with 1 nM R1881 or ethanol for 24 hrs. Luciferase activity relative to Renilla was calculated and then normalised to cells with 0 ng Nodal and treated with R1881. Average values from 3 experiments are plotted with SEM. There was a significant decrease in luciferase activity with Nodal co- transfection (One Way Anova, n=3, * P<0.05, ** P<0.01). B – QRT-PCR comparing gene expression in LNCaP cells stably transfected with Nodal or vector only and cultured in media containing 10% FCS. There was a significant decrease in KLK2, 3 and 15, TMPRSS2 and NKX3.1 expression (t test, n=3, * P<0.05, ** P<0.01, *** P<0.001).

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control and then treated for 24 hrs with 1 nM R1881. In the absence of Nodal, there was a 5-fold increase in KLK3 reporter activity (Figure 5.9A). Co-transfecting increasing amounts of the Nodal construct significantly attenuated KLK3 luciferase activity in R1881 treated cells. The endogenous expression of androgen-regulated genes was compared between the LNCaP cells stably transfected with Nodal or vector only and grown in the presence of total serum. There was a significant decrease in the expression of androgen-regulated kallikrein genes, KLK2, 3 and 15 in the Nodal-expressing cell line (Figure 5.9B). The decrease in KLK4 mRNA levels was not statistically significant (P=0.19). TMPRSS2 and NKX3.1, prototypical androgen-regulated genes, were also significantly down-regulated. Interestingly, there was a trend of decreased AR (P=0.08) but increased KLK14 (P=0.15) expression in the LNCaP cells transfected with Nodal. There was no difference in Cripto expression (P=0.52). Altogether, these results demonstrate that Nodal antagonises AR signalling in prostate cancer cells.

5.3.9 Cleavage of Nodal by Kallikrein-Related Serine Peptidases Proteolytic maturation of pro-Nodal determines the activity and stability of the ligand (Le Good et al., 2005). The role of furin and PACE4 in Nodal activation has not been confirmed in human cells or the tumour microenvironment. Therefore, RT-PCR was used to evaluate furin and PACE4 expression in prostate cancer and melanoma cell lines and hESCs. All cells expressed at least one of these genes while most expressed both (Figure 5.10A). Furin and PACE4 target an RXRR motif in the Nodal pro-domain that is highly conserved between species. The sequence of this motif is RHRR in humans (Figure 5.10B). As serine proteases with trypsin-like specificity, several members of the kallikrein family are able to cleave sequences similar to that of the Nodal cleavage site. These include KLK4 and KLK14 which have a strong preference for arginine in the P1 position (Felber et al., 2005, Matsumura et al., 2005, Borgono et al., 2007a). Given the increasing abundance of kallikreins in the prostate cancer microenvironment with the loss of glandular architecture, it is possible that they are pathophysiological activators of pro-Nodal. To test this hypothesis KLK2, 3, 4, 6 and 14 were co-transfected with Nodal into COS-1 cells. Nodal undergoes minimal processing by endogenous proteases in this cell line (Constam and Robertson 1999, Yan et al., 2002). For each kallikrein, catalytically inactive mutants with an alanine substitution for the catalytic serine were used as negative controls. After 48 hrs, the serum free conditioned media was collected to assess Nodal cleavage. Figure 5.11A summarises the results for KLK4 which are representative of all the kallikreins tested. A non-specific band was detected with the Flag antibody in the vector only control and pro-Nodal was detected as a band of approximately 42 kDa. The Flag tag is attached to the C-terminus of Nodal so the mature form should also be immunoreactive. However, no mature Nodal was observed, even in cells transfected with wild-type KLK4. Since mature Nodal is rapidly degraded, it may be

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Figure 5.10. The Nodal Pro-Domain Cleavage Site and Proprotein Convertase Expression A – RT-PCR of PACE4 and furin in prostate and melanoma cell lines and H9 hESCs. The housekeeping gene, HRPT1, was used to compare cDNA concentrations. NTC = no template control. B – Alignment of the Nodal pro-peptide cleavage site showing the high conservation among species. The arrow shows the cleavage site after the RXRR motif. P and P’ residues of the cleavage motif are labelled.

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Figure 5.11. Transfected KLK4 Does Not Activate Nodal in Cos-1 Conditioned Media A – Cos-1 cells were co-transfected with pre-Nodal and wild-type (WT) or mutant (MT) pre- pro-KLK4. Cells were transfected with vector only as a negative control. Serum-free conditioned media was collected after 48 Hrs. No mature Nodal or decrease in pro-Nodal was detected in Western blots with an anti-Flag antibody. The V5 antibody was used to confirm equal levels of wild-type and mutant KLK4 in the conditioned media. B – Western blots of whole cell lysates verify that there were equivalent amounts of Nodal and KLK4 between transfections.

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difficult to detect. Yet there was no diminution in the amount of pro-Nodal cells transfected with wild-type KLK4 compared with mutant KLK4 and Nodal alone. Western blots of conditioned media and whole cell lysates confirmed that wild-type and mutant KLK4 were expressed at similar levels (Figure 5.11A & B). Furthermore, Nodal expression in whole cell lysates was also equivalent between different transfections (Figure 5.11 B). In summary, there was no detectable cleavage of pro-Nodal by KLK2, 3, 4, 6 or 14 using this approach.

It is possible that the lack of pro-Nodal cleavage in co-transfection experiments was due to the kallikreins being inactive under these conditions. Accordingly, when the serum free conditioned media samples were spiked with FCS, the transfected kallikreins did not bind to the protease inhibitors present in serum (data not shown). This would have required enzyme activity and suggests that the transfected kallikreins were inactive or otherwise inhibited. This problem could be circumvented by using recombinant kallikrein proteins that are known to be proteolytically active. Our laboratory has noted that cell culture media inhibits the activity of recombinant kallikreins (personal communication Ms Satomi Okano, QUT). Therefore, pro-Nodal first had to be purified from conditioned media samples as only mature recombinant Nodal is commercially available. Nickel affinity purification was used to extract pro-Nodal from the conditioned media of stably or transiently transfected COS-1 cells. A typical purification is shown in Figure 5.12A with pro-Nodal present in the first two elution fractions. The amount of Nodal in each batch was quantified using a standard curve of commercially available mature Nodal (Figure 5.12B).

KLK3 purified from seminal plasma and recombinant KLK4 and KLK14 were chosen for enzyme substrate assays. All enzymes were active against peptide substrates (personal communication Ms Janet Reid and Dr Scott Stansfield, QUT). No trypsin-like activity was detected in KLK3 samples confirming that closely related kallikreins such as KLK2 had not been co-purified from seminal plasma. Before KLK3, 4 and 14 were incubated with pro-Nodal, their proteolytic activity against other protein substrates was verified. The ectodomain of E- cadherin is efficiently cleaved by several kallikreins (personal communication Drs Astrid Whitbread and Tara Veveris-Lowe, QUT and Johnson et al., 2007)). Consistent with these observations, active KLK4 and KLK14 degraded the Fc-fused E-cadherin within 30 mins (Figure 5.13A). There was no degradation in the absence of proteases or for the thermolysin and phosphoramidon (T/P) control. The E-cadherin ectodomain was also cleaved when pro-KLK14 but not pro-KLK4 was added, suggesting that KLK14 is able to auto-activate under certain conditions. Proteolysis of E-cadherin:Fc proceeded more slowly for KLK3 than KLK4 and KLK14, requiring overnight incubation for complete degradation (Figure 5.13A).

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Figure 5.12. Purification and Quantifaction of Pro-Nodal A – Western blot of elutions, flow through and washes of a pro-Nodal purification using anti- Flag primary antibody. Whole cell lysate from Cos-1 cells transiently transfected with Nodal was used as a positive control. B – Western blot of purified pro-Nodal from Cos-1 conditioned media compared with a standard curve of recombinant mature Nodal from E. coli. The anti-Flag primary antibody cross-reacted with BSA in the mature Nodal samples.

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Figure 5.13. KLK3, KLK4 and KLK14 Cleave Protein Substrates A – Western blot of E-cadherin:Fc fragments using an anti-E-cadherin antibody. Digests containing 100 ng E-cadherin:Fc and 30 ng Kallikrein were incubated at 37 ºC for 0.5 hrs, 2 hrs or overnight (ON). B – Silver stained gels of fibronectin digests containing 500 ng substrate and 25 ng KLK4 and KLK14 or 50 ng KLK3. Samples were incubated at 37 ºC for 0.5 hrs, 2 hrs or overnight. KLK3, 4 and 14 all degraded both substrates. T/P = thermolysin and phosphoramidon controls, Untx = untreated control.

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This concurs with a previous report that KLK3 is a relatively inefficient enzyme (Coombs et al., 1998). Fibronectin is also a substrate of many kallikreins including KLK3 and KLK14 (Webber et al., 1995, Gallardo-Williams et al., 2003, Borgono et al., 2007b, Rajapakse and Takahashi 2007). Accordingly, KLK4 and KLK14 degraded fibronectin within 30 mins, while overnightincubation was required for KLK3 (Figure 5.13B). Once again, there was no degradation in the absence of kallikreins or in the T/P control. These experiments confirm that the KLK3, 4 and 14 used in this study are enzymatically active against protein substrates.

Pro-Nodal was next combined with recombinant kallikreins to investigate whether it might also be a substrate. A 1:4.8 molar ratio of KLK4 and KLK14 to pro-Nodal and a 1:4.8 molar ratio of KLK3 to pro-Nodal were incubated overnight at 37 ºC. High molar ratios were used to resolve whether each kallikrein had any ability to cleave pro-Nodal. It should also be noted that the ratios are to total rather than active kallikrein protein. Under these conditions both KLK4 and KLK14 degraded pro-Nodal whereas KLK3 did not (Figure 5.14A). Cleavage was specific for KLK4 and KLK14 because there was no degradation of untreated pro-Nodal or with the T/P control. Given that KLK4 and KLK14 have similar substrate specificity, further experiments were conducted with KLK4 since pre-activated batches of known activity were available. This allowed accurate calculation of molar ratios between pro-Nodal and active enzyme. To investigate the affinity of KLK4 for pro-Nodal, different molar ratios were combined and incubated at 37 ºC overnight. KLK4 only cleaved pro-Nodal when present at a 1:1 molar ratio (Figure 5.14B). Notably, a 23 kDa degradation product similar to the expected size of mature Nodal was observed in these experiments (Beck et al., 2002). Silver staining revealed that the pro-Nodal purifications contained contaminants (data not shown). The ratio of pro-Nodal to total protein was quantified as 1:125. This means that the ability of KLK4 to cleave pro-Nodal may have been underestimated. Therefore, proNodal was further enriched through immunoprecipitation with the rabbit anti-human Nodal antibody. The ratio of pro-Nodal to total protein in these samples was approximately 1:3. KLK4 was slightly more efficient at cleaving pro-Nodal in these experiments with some cleavage at a 1:10 molar ratio of KLK4 to pro-Nodal (Figure 5.14C). An intermediate cleavage product of approximately 45 kDa was detected in these experiments representing partial cleavage of the pro-domain. These results demonstrate that kallikreins with trypsin-like specificity are able to cleave pro-Nodal. However, since this only occurs at high molar ratios, the kallikreins are unlikely to be physiological regulators of pro-Nodal in the tumour microenvironment.

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Figure 5.14. Cleavage of Pro-Nodal by Prostatic Kallikreins A – Nodal (4 ng) was degraded by 2 ng KLK4 and KLK14 but not 8 ng KLK3. B – KLK4 cleaved Nodal at 1:1 but not higher molar ratios of pro-Nodal to active protease. The ~23 kDa product is the expected size of mature Nodal. The ratio of Nodal to total protein in the purifications used in this experiment was 1:125. C – Digests were repeated with immunoprecipitated pro-Nodal with a ratio of 1:3 Nodal to total protein. KLK4 cleaved Nodal at 1:1 and 1:10 molar ratios with an intermediate cleavage product of ~44 kDa. There was no cleavage at 1:100 and 1:1000 molar ratios. All digests were performed overnight at 37 ºC. Western blots were probed with the anti-Flag primary antibody. Arrows indicate the pro-Nodal band and the expected size of mature Nodal.

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5.4 Discussion Prostate development, homeostasis and tumour progression are guided by dynamic interactions between prostate epithelial cells and the microenvironment. Furthermore, as prostate cancer cells develop into tumours, invade their local surroundings, extravasate into circulation, form micrometastases and finally form secondary tumours, they must adapt to their varied surroundings. Investigating how cancer cells respond to different microenvironments may provide a better understanding of important steps in tumour progression.

Recent studies have used conditioned matrices to model different aspects of the microenvironment (Seftor et al., 2005, Postovit et al., 2006, Seftor et al., 2006, Khalkhali-Ellis and Hendrix 2007, Postovit et al., 2008b). This novel three-dimensional technique can be used to investigate interactions between many different cell types, such as normal and malignant cells or tumour cells and stroma from their primary tissue or metastatic niche. In this study, the CMTX model was first used to examine interactions between different sublines of DU145 cells. Vasculogenic mimicry was used to measure changes in plasticity because it requires matrix remodelling and heterotypic interactions between different populations of cancer cells (Sharma et al., 2002b). The ECM-rich vasculogenic networks are thought to aide in the perfusion of oxygen and nutrients to tumour cells and have been identified in various malignancies including melanoma, breast, ovarian and prostate cancer (Maniotis et al., 1999, Hendrix et al., 2002, Liu et al., 2002a, Sharma et al., 2002b, Sood et al., 2002, Shirakawa et al., 2003). Moreover, a previous study has shown increased network formation by poorly aggressive uveal and cutaneous melanoma cells exposed to CMTX from metastatic melanoma cell lines (Seftor et al., 2006). When DU145-E and DU145-F sublines were separately cultured on collagen I, collagen IV and laminin matrices, they did not exhibit vasculogenic mimicry, unlike parental DU145 cells or a 1:1 mixture of the two sublines. However, DU145-E cells exposed to DU145-F CMTX successfully formed back to back loops indicative of vasculogenic networks. This result confirmed the importance of cell-cell signalling for vasculogenic mimicry and demonstrated that DU145 cells are responsive to inductive cues within conditioned matrices. Interestingly in further experiments, neither primary (PrEC) nor immortalised (MLC8891) normal prostate epithelial cells responded to DU145 or PC-3 CMTX (data not shown). Unlike normal melanocytes cultured on metastatic melanoma CMTX (Seftor et al., 2005), there was no change in morphology, migration, invasion or expression of candidate genes. It is possible that the cells were altered in other ways that were not detected, or that only transformed prostate cells are affected by this model.

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Given that conditioned matrices can enhance the plasticity of tumour cells, we hypothesised that they could also be used to attenuate the aggressiveness of prostate cancer cells. Indeed, DU145 and PC-3 cells cultured on hESC CMTX were unable to undergo vasculogenic mimicry and had decreased clonogenicity in soft agar assays. These experiments were based on the notion that tumour progression is a caricature of development, except that adult tissues lack the regulatory factors that guide cell fate in embryos (Pierce and Speers 1988). The hESC CMTX may have restored the balance to dysregulated developmental signalling pathways expressed by DU145 and PC3 cells. A number of other studies have also shown that embryonic microenvironments, previously termed “embryonic fields”, can reprogram tumour cells to a less aggressive phenotype. For instance, melanoma cells injected into zebrafish, chick or mouse embryos have reduced tumourigenicity and behave either like differentiated melanocytes or their neural crest cell precursors (Gerschenson et al., 1986, Lee et al., 2005, Kulesa et al., 2006). In addition, neuroblastoma cells form fewer tumours in vivo when they are co-implanted with neurula stage mouse embryos compared with sections of normal adult liver (Podesta et al., 1984). Perhaps the most dramatic example is that mouse blastocysts injected with teratocarcinoma cells and then transferred back into pregnant mice develop normally with the tumour cells integrated throughout different tissues (Brinster 1974, Mintz and Illmensee 1975). With respect to the prostate, when cores of Dunning and Noble rat prostate tumours are recombined with UGM or seminal vesicle mesenchyme (SVM), they recanalise and form acinar ductal structures with secretory activity (Chung et al., 1990, Hayashi et al., 1990, Hayashi and Cunha 1991). Similarly, mouse NeoTag prostate cancer cells form dense undifferentiated masses when transplanted into the rat renal capsule alone, but develop into glandular structures when mixed with UGM (Wang et al., 2006b). However in this instance, UGM did not prevent tumour formation. These observations suggest that embryonic cues are powerful regulators of many different tumour types, from adenocarcinomas, such as prostate cancer, to stem-cell rich embryonal tumours from the testes. Many of these studies have exposed tumour cells to the foetal microenvironments of their developmental precursors; melanoma cells were injected into embryonic skin and prostate cancer cells were recombined with UGM. The hESC CMTX model is a more extreme version of these experiments and demonstrates that melanoma, breast and prostate cancer cells are responsive to even earlier developmental cues.

Together, these studies showing that embryonic microenvironments reduce the aggressiveness of tumour cells are a fascinating proof of principle. The next step is to uncover the molecular mechanisms underlying these changes in the hope they offer novel therapeutic targets. A recent study used the hESC CMTX model to identify such factors and showed that hESC-derived Lefty reduces the clonogenicity and tumourigenicity of C8161 melanoma and MDA-MB-231 breast cancer cells (Postovit et al., 2008b). These effects are due to Lefty inhibition of Nodal, an

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embryonic signalling molecule that is re-expressed by aggressive tumour cells (Topczewska et al., 2006). Significantly, Nodal was down-regulated in DU145 cells exposed to hESC CMTX suggesting that Lefty was also acting on this cell line. This was the first observation of Nodal expression in prostate cancer. Increased Nodal or Cripto expression has been reported in melanoma and cancers of the breast, colon, lung, ovary, endometrium, cervix, gall bladder, pancreas and stomach (Saloman et al., 2000, Topczewska et al., 2006).Yet, little is known about the expression of the Nodal axis in the prostate. One immunohistochemical study was unable to detect Cripto expression in prostate cancer specimens (Okajima et al., 1997). However, this report also failed to observe staining in other malignancies that are known to up-regulate Cripto such as bladder and testicular cancer (Tabibzadeh et al., 1997, Byrne et al., 1998). More recently, Cripto blocking antibodies were shown to inhibit the growth of LNCaP, PC-3 and DU145 cells in vitro, implying that the Nodal axis may be important for the growth and survival of prostate cancer cells (Xing et al., 2004). Therefore, the expression and function of the Nodal axis in prostate cancer warranted further investigation.

The expression of key factors involved in Nodal signalling was first characterised in prostate cell lines. Nodal protein was only detected in DU145 and PC-3 cells whereas Cripto was expressed by all cell lines. In contrast Lefty mRNA was absent in prostate cell lines but expressed in hESCs which were used as a positive control. Previous reports have confirmed the expression of activin type II receptors and Smad2/3 in most of these cell lines (Yang et al., 2005a, Lu et al., 2007). In this study, Nodal expression was also examined in prostate tissue using immunohistochemistry and shown to increase in Gleason grade 3 and 4 cancer compared with benign glands. This is consistent with previous reports of greater Nodal staining in poorly differentiated breast cancer and melanoma samples (Topczewska et al., 2006, Postovit et al., 2008b).

Other factors that modulate Nodal signalling remain to be characterised in prostate cancer. In mouse embryos Nodal signals through Cripto to establish the anterior-posterior axis but then through Cryptic, a closely related member of the EGF-CFC receptor family, to direct left-right symmetry (Gaio et al., 1999, Yan et al., 1999). Cryptic mutations cause laterality defects such as congenital heart malformation in humans (Bamford et al., 2000). Cryptic expression in tumour specimens has not been reported; however it is not present in adult tissues or embryonal carcinoma cells of the mouse (Shen et al., 1997). In addition, although Lefty is not produced by prostate cells, the expression of another Nodal antagonist, Cerberus, has not been tested. Cerberus inhibits Nodal, BMP and Wnt signalling in anterior endoderm to allow correct neural development (Piccolo et al., 1999, Belo et al., 2000, Bell et al., 2003). Although, Cerberus is

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expressed in hESCs and a subpopulation of C8161 melanoma cells, its levels in patient tumour tissues have not been reported (Besser 2004, Strizzi et al., 2008).

Even though some factors in the Nodal axis require further characterisation, the expression of key molecules, such as Cripto, suggest that prostate cancer cells may be responsive to Nodal signalling. Indeed, recombinant Nodal triggered Smad2 phosphorylation in DU145 cells which produce endogenous Nodal as well as LNCaP cells that lack Nodal expression. These results imply that Nodal is able to act in trans on prostate cancer cells expressing Cripto. Intriguingly, Cripto can also function in a non-cell autonomous manner in embryos and tumour cell lines (Yan et al., 2002, Bianco et al., 2003, Chu et al., 2005). The soluble form of Cripto lacking its GPI anchor is able to initiate Nodal-dependent signalling through the activin receptors and rescue the development of Cripto null embryos (Chu et al., 2005). Significantly, there are increased levels of soluble Cripto in the sera of breast and colon cancer patients (Bianco et al., 2006). Therefore, a subpopulation of tumour cells producing Nodal and Cripto could signal to adjacent cells as long as they express ActRIIA/B and Alk4/7. This would be exacerbated by the lack of Lefty feedback inhibition. The potential paracrine actions of Nodal and soluble Cripto in prostate cancer warrant further investigations.

Nodal successfully stimulates Smad2 phosphorylation in prostate cancer cells, but it is not yet known through which combination of ActRII and Alk receptors this signal is propagated. Of the type I serine threonine kinase receptors, Alk4 is the main candidate. It is expressed in prostate cancer cell lines, xenografts, and tissues, whereas Alk7 was not detected in a Northern blot of prostate tissue extracts (van Schaik et al., 2000, Bondestam et al., 2001). ActRIIA and ActRIIB are also expressed in all prostate cell lines and tissue samples tested (van Schaik et al., 2000, Yang et al., 2005a). However, LNCaP, DU145, 22RV1 and LAPC-4 prostate cancer cells have single nucleotide deletions in the kinase domain of ActRIIA that diminish its ability to initiate downstream signalling (Rossi et al., 2005). LNCaP and DU145 cells are heterozygous for these mutations, 22Rv1 and LAPC-4 are homozygous, while PC-3 cells express wild-type ActRIIA. The prevalence of such mutations in prostate cancer patients is unknown. Nevertheless, in prostate cancer cell lines ActRIIB would be required for maximal Nodal signal transduction, likely in combination with Alk4.

Higher concentrations of Nodal were needed to induce Smad phosphorylation in DU145 cells compared with TGFβ1 which binds to TGFβ receptor I (TGFRI/Alk5) and TGFRII (Shi and Massague 2003). These results are consistent with other studies suggesting that Nodal signalling is of lower potency and efficiency than related ligands such as TGFβ1 and activin. One study estimated that Nodal, with an EC50 of 14 nM (~150 ng/mL), is 250 times less potent than activin A with an EC50 of 60 pM (~0.5ng/mL) (Kelber et al., 2008). The amount of rNodal added to

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prostate cancer cells was similar to doses used by Kelber and co-workers and lower than other studies with hESCs and pancreatic cells (Kumar et al., 2001, Vallier et al., 2005, Zhang et al., 2008). The ActRIIA mutations may partly account for the modest activity of Nodal signalling, particularly if the mutant receptor binds Nodal but fails to properly initiate downstream Smad phosphorylation. Notably, LNCaP cells are responsive to Nodal but not TGFβ1, because TGFRI and TGFRII are not expressed due to promoter methylation (Zhang et al., 2005, Zhao et al., 2005b). The intensity of Nodal signalling is also determined by the amount of cell surface or soluble Cripto. In cell culture experiments where the amount of Nodal is kept constant, downstream signalling increases with greater amounts of Cripto until a saturation point where the co-receptor is contained within every ActRIIA/B-Alk4/7 complex (Kelber et al., 2008). Although Cripto is essential for Nodal activation of ActRIIA/B and Alk4/7, it reduces the potency and efficiency of the receptor complex (Kelber et al., 2008). This explains the modest response of cells to Nodal compared with other TGFβ ligands. Since activin binds to the same receptors as Nodal, ActRIIA/B and Alk4, high levels of Cripto attenuate activin signalling (Gray et al., 2003, Kelber et al., 2008). Indeed, activins are generally more potent than Nodal because they do not require Cripto (Kumar et al., 2001, Vallier et al., 2005, Kelber et al., 2008, Zhang et al., 2008). Cripto also antagonises TGFβ, in this case by directly binding to the ligand and blocking its association with Alk5 (Gray et al., 2006). Therefore, depending on the relative expression of Nodal, activins and TGFβ, Cripto can either enhance or diminish Smad2/3 phosphorylation.

The lower intrinsic activity of Nodal compared with other TGFβ ligands does not mean that it is less biologically relevant. In embryos Nodal acts a morphogen that not only acts where it is expressed, but at a distance in a concentration-dependent manner (Chen and Schier 2001, Meno et al., 2001). The formation of endoderm requires higher levels of Nodal, whereas mesoderm still develops at lower concentrations (Schier et al., 1997, Thisse et al., 2000). Downstream gene expression also varies with the activity of the Nodal axis; brachyury expression requires robust signalling, while goosecoid is stimulated by more modest levels (Gritsman et al., 2000). It is likely that the effect of Nodal on tumour cells will also be concentration-dependent. Furthermore, even though Nodal stimulates Smad2/3 phosphorylation like activin and TGFβ, the downstream consequences may be quite distinct due to differences in the intensity of the signal.

Nodal signalling in prostate cancer cells has functional effects, particularly on anchorage- independent growth which is considered to be a measure of the stem-like characteristics of tumour cells (Hamburger and Salmon 1977, Buick and Pollak 1984). LNCaP cells stably transfected with Nodal had increased clonogenicity in soft agar assays. This is the inverse response compared with DU145 cells exposed to hESC CMTX in which Nodal was down-

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regulated. The results for PC-3 cells were different; there was no change in clonogenicity with Nodal transfection and no decrease in Nodal expression when the cells were cultured on hESC CMTX. Both observations suggest that PC-3 cells are poorly responsive to Nodal even though it is expressed in this cell line. In DU145, MDA-MB-231, and C8161 cells, hESC-derived Lefty blocked the positive feedback loop through which Nodal up-regulates its own transcription. Nodal levels were not affected in PC-3 cells where endogenous Nodal expression seems to be uncoupled from the lack of Nodal signalling. PC-3 cells are also unresponsive to activin, although this is most likely due to over-expression of membrane-bound follistatin which inhibits activin but not Nodal activity (Dalkin et al., 1996, McPherson et al., 1999, Harrington et al., 2006). Despite these idiosyncrasies, the clonogencity of PC-3 cells was still abrogated by hESC CMTX although, unlike DU145 cells, there was no change in invasion. This is in accordance with previous suggestions that there are other tumour suppressive factors in hESC CMTX besides Lefty (Postovit et al., 2008b). PC-3 cells may be an ideal model to help identify these molecules.

Another consequence of Nodal signalling in prostate cancer cells is antagonism of AR activity. Interactions between the TGFβ superfamily and AR axes are well recognised. Correct prostate development during embryogenesis relies on a balance between androgens, which stimulate growth and branching morphogenesis, and activin, TGFβ1 and TGFβ2, which inhibit these processes (Ball and Risbridger 2003). Androgens are vital for the survival of epithelial cells within the adult prostate, whereas TGFβs cause apoptosis and involution upon castration (Huggins and Hodges 1941, English et al., 1987, Kyprianou and Isaacs 1988, Martikainen et al., 1990). Furthermore, many factors in the TGFβ superfamily and downstream pathways are regulated by androgens. Activin βB, TGFβ1-3, TGFRI and II, Smads 2, 3, and 4 are all repressed by androgens (Nishi et al., 1996, Lucia et al., 1998, Brodin et al., 1999, Chipuk et al., 2002, Song et al., 2008). In contrast, androgens stimulate activin A, ActRIIA, BMP7 and BMP receptor IIB (Alk6) expression (Risbridger et al., 1996, Ide et al., 1997, Thomas et al., 1998, Al- Omari et al., 2005). TGFβ ligands can also modulate AR. BMPs inhibit AR activity through Smad1, which acts as a co-repressor in prostate cancer cells (Qiu et al., 2007). There is also a direct interaction between Smad3 and AR. However, studies disagree whether this enhances or represses AR activity, even within the same cell lines (Hayes et al., 2001, Kang et al., 2001, Kang et al., 2002). This suggests that the downstream effects of the AR-Smad3 interaction are highly context-dependent and may explain why androgen-regulated gene expression is stimulated by activin but inhibited by TGFβ1 (Gerdes et al., 1998, Hayes et al., 2001, Fujii et al., 2004).

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To investigate the role of Nodal in AR signalling, the expression of endogenous androgen- regulated genes in stably transfected LNCaP cells was examined. KLK2, 3, 4, TMPRSS2 and NKX3.1 were all down-regulated. There was also reduced AR expression. The antagonism of AR signalling by Nodal was confirmed with luciferase assays using a KLK3 reporter construct and increasing amounts of transiently transfected pre-pro-Nodal. A dose responsive decrease in reporter activity was observed. It remains to be determined whether androgens have a reciprocal effect on Nodal signalling. As noted, Smad 2, 3 and 4 are up-regulated but ActRIIA is down- regulated upon castration. Preliminary experiments showed that Cripto expression was not altered by R1881 treatment or hormone deprivation of LNCaP cells (data not shown). The effect of androgens on Nodal expression has not yet been tested because it is only expressed in DU145 and PC-3 cells which lack AR. Nevertheless, results so far demonstrate that Nodal attenuates AR activity. In future studies it will be worthwhile to compare the expression and activity of the Nodal axis in androgen-responsive, hormone-deprived and castrate-resistant stages of prostate cancer.

Nodal signalling depends on the amount of mature ligand. Therefore, proteases that cleave the Nodal pro-peptide regulate the activity of the Nodal axis. In mouse embryos, two serine proteases of the proprotein convertases family, furin and PACE4 activate Nodal by cleaving a RQRR motif in the pro-domain. The sequence of this motif is RHRR in humans. Unlike many other proprotein convertase substrates that are activated in the trans-Golgi network, Nodal is processed extracellularly (Beck et al., 2002, Seidah et al., 2008). This allows Nodal to act as a morphogen, diffusing to distant cells in its inactive pro-form before proteolytic maturation and signalling. We hypothesised that kallikrein-related serine peptidases may also be able to activate pro-Nodal given their abundance in the prostate cancer microenvironment and similar ability to cleave after arginine residues. Intriguingly, there is also an overlap between kallikrein and Nodal expression in embryonic cells. For example, KLK1 is expressed in mouse embryos from the onset of gastrulation (Chan et al., 1999) and KLK4, 5, 8, 13 and 14 are all expressed in hESCs (Appendix 3). However, KLK3, 4 and 14 all proved to be inefficient at cleaving pro-Nodal in vitro. This was not surprising for KLK3 which has a preference for cleaving after lysine and tyrosine residues (Coombs et al., 1998). In contrast, KLK3 is able to activate TGFβ2, albeit via cleavage of the latency-associated peptide that regulates the activity of TGFβs, but not Nodal (Killian et al., 1993, Dallas et al., 2005). KLK4 and KLK14 both cleaved pro-Nodal, but only at high molar ratios. It is important to distinguish between activation and degradation of pro-Nodal. Occasionally, KLK4 and KLK14 generated cleavage products of the expected size of mature Nodal. In other instances there was complete clearance of Nodal protein. This suggests that KLK4 and KLK14 have low affinity for the RHRR activation motif. Indeed, phage display and positional candidate screening of synthetic-combinatorial libraries indicate that KLK4 and

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KLK14 rarely cleave peptides with arginine residues in the P2 and P4 position (Felber et al., 2005, Matsumura et al., 2005, Borgono et al., 2007a). Although KLK4 and KLK14 were able to degrade pro-Nodal in vitro, this only occurred with high concentrations of enzymes during overnight incubations. In comparison, KLK4 and KLK14 have been shown to degrade other substrates within 4 hrs at molar ratios of 1:1000 (Dr Scott Stansfield, QUT, personal communication and Borgono et al., 2007b). These observations imply that Nodal is unlikely to be a major substrate of kallikreins in the prostate microenvironment.

The role of Nodal in prostate cancer progression in vivo is still uncertain. Its effects are likely to be highly context-dependent. Smads must complex with other transcription factors to activate downstream gene expression. This means that the transcriptional targets and biological consequences of Smad signalling are quite variable (Miyazawa et al., 2002). For example, TGFβs, which activate Smad2/3, and BMPs, which trigger Smad1/5 phosphorylation, both inhibit the proliferation of normal prostate epithelial cell but induce EMT in malignant cell lines (Yang et al., 2005a, Ao et al., 2007). Cancer cells are able to evade the cytostatic effects of Smads but still harness the tumour promoting properties of TGFβ ligands. Hyper-activation of the PI3K/Akt pathway in cancer inhibits the FOXO transcription factors which usually interact with Smads and stimulate the expression of anti-proliferative target genes (Massague and Gomis 2006). The downstream effects of Smads are also determined by the strength and duration of the signal. In HaCat keratinocytes TGFβ induces growth arrest after 14 hrs of continuous treatment but not 12 hrs (Nicolas and Hill 2003).

Predicting the context-dependent effects of Nodal is further complicated by the Nodal- independent actions of Cripto. The PI3K/Akt, Src and ras/raf/MAPK pathways are all activated by Cripto independent of Nodal and Alk4/7 and ActRIIA/B (De Santis et al., 1997, Kannan et al., 1997, Ebert et al., 1999, Bianco et al., 2003). As previously noted, Cripto is also able to attenuate activin and TGFβ signalling. There has been extensive research into the role of Cripto in cancer progression. It promotes proliferation, migration, invasion, EMT, angiogenesis and tumourigenesis (Strizzi et al., 2005). In a few cases the Nodal-dependent and independent changes have been dissected. For example, Cripto-induced migration and differentiation of endothelial cells occurs via PI3K and Src but not Nodal or MAPK (Bianco et al., 2005). Cripto inactivation with antisense oligonucleotides or blocking antibodies has been shown to inhibit the growth of a variety of tumour models including PC-3, DU145 and LNCaP prostate cancer cells (Ciardiello et al., 1994, Adkins et al., 2003, Normanno et al., 2004, Xing et al., 2004). It is unclear whether this is due to disruption of Nodal, other signalling cascades or both.

Understanding how Nodal is transcriptionally regulated may help clarify its role in prostate cancer progression by placing it in the context of upstream pathways. Nodal expression in

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embryos is highly context-dependent (Schier 2003). The most consistent activator of Nodal transcription is Nodal itself through downstream binding of Smad2 and FoxHI to sites within intron 1 (Adachi et al., 1999, Norris and Robertson 1999, Norris et al., 2002). This suggests that a subpopulation of tumour cells producing Nodal could trigger further expression in adjacent cells. β-catenin also regulates Nodal expression in zebrafish and xenopus embryos during mesendoderm induction (Agius et al., 2000, Whitman 2001, Dougan et al., 2003). Furthermore, inhibition of GSK3β activity prevents differentiation and maintains Nodal expression in hESCs, perhaps through β-catenin (Besser 2004). Cripto expression is also stimulated by β-catenin in mouse embryos and colon cancer cell lines (Morkel et al., 2003). This suggests that β-catenin is a good candidate for stimulating the expression of the Nodal axis in prostate cancer. In preliminary experiments there was no change in Nodal or Cripto expression in DU145 cells treated with SB216763, a GSK3β inhibitor, and transfected with β-catenin or control siRNA (data not shown). However, as the cells were only treated with SB216763 for 8 hrs in these experiments, longer time-courses should be conducted. The Notch pathway may also regulate Nodal expression. Notch signalling stimulates Nodal transcription in the node during left-right axis formation in embryos (Krebs et al., 2003, Raya et al., 2003). The Notch pathway is also up- regulated during prostate development and tumourigenesis and is expressed in all prostate cell lines used in this study (Leong and Gao 2008). Curiously, Nodal mRNA was detected at similar levels in all prostate cell lines even though Nodal protein was only observed in DU145 and PC-3 cells (data not shown). This implies that Nodal is also subject to post-transcriptional regulation. Indeed, Nodal, Lefty and ActRIIA levels are all tightly regulated by miRNAs in embryos (Choi et al., 2007, Martello et al., 2007). Therefore, Nodal expression is likely regulated by multiple mechanisms including embryonic pathways that are reactivated during prostate cancer progression.

The original premise of this study was that prostate cancer cells are susceptible to regulatory cues from an embryonic microenvironment because they commandeer and corrupt developmental signalling pathways. Many factors that guide the development of the foetal prostate are reactivated in prostate cancer progression including the Hedgehog, Wnt, Notch, FGF and Sox9 pathways (Karhadkar et al., 2004, Verras and Sun 2006, Leong and Gao 2008, Schaeffer et al., 2008, Wang et al., 2008c). This raises the question whether Nodal is also involved in prostate growth and morphogenesis. Recombination experiments have recently shown that UGM can direct hESCs to differentiate into prostate ducts (Taylor et al., 2006). The UGM must support Nodal signalling in the early stages of this process because it is required for specification of embryonic stems cells to definitive endoderm (Takenaga et al., 2007, Smith et al., 2008). It is also possible that Nodal influences later steps in prostate development. BMPs, TGFβ and activins all inhibit branching morphogenesis in the prostate (Prins and Putz 2008). Yet

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in breast, Nodal and Cripto can stimulate ductal morphogenesis, unlike other the members of the TGFβ family (Kenney et al., 2004). This suggests that, if Nodal has a role in prostate development, it may be different from other TGFβ ligands.

In summary, the data presented in this chapter indicate that the plasticity of prostate cancer cells is regulated by the microenvironment. The conditioned matrix model can be used to enhance or attenuate the aggressive phenotype of prostate cancer cell lines. Through these experiments Nodal was identified as a novel factor that is up-regulated in prostate cancer progression and has a functional role in activating the Smad pathway, modulating AR activity and increasing clonogenicity. The ability of kallikreins to cleave pro-Nodal was also investigated, showing that they are unlikely to be physiological activators of Nodal activity.

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6Chapter 6: General Discussion

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Prostate cancer is a significant male health issue. It accounts for 25% of cancer diagnoses and 10% of cancer-related deaths (Jemal et al., 2008). Men with localised prostate cancer have a 5 year survival rate of almost 100%, but endure side effects from therapeutic interventions. A subset of tumours that are recurrent or have already invaded beyond the prostate at the time of diagnosis are more challenging to treat and are often fatal. A better understanding of the cellular and molecular mechanisms underlying prostate cancer progression is required to improve patient outcomes.

The strategies for diagnosis and treatment of prostate cancer emphasise key aspects of prostate cancer biology. Advanced tumours are targeted by hormone deprivation therapy, because prostate cancer is initially an androgen-dependent disease (Huggins and Hodges 1941). The Gleason grading system classifies the aggressiveness of tumours based on the morphology of glands, because prostate cancer involves expansion and dedifferentiation of epithelial cells (Gleason 1977). The focus of this project was the two themes of androgen regulation and cellular differentiation, and the relationships between them.

6.1.1 Androgens and Kallikrein Expression in Prostate Cancer Androgens are essential for the formation and function of the prostate. The AR stimulates budding of the foetal prostate, branching morphogenesis, growth and maturation during puberty and secretion of seminal plasma proteins in adulthood. The differing roles of mesenchymal and epithelial AR balance the actions of androgens during prostate development and homeostasis (Cunha and Lung 1978, Donjacour and Cunha 1993). The AR is also crucial in the development and progression of prostate cancer. Whereas AR inhibits proliferation and enhances the differentiation of normal prostate epithelial cells, it increases the tumourigenicity of transformed prostate epithelial cells (Berger et al., 2004, Xin et al., 2006). By co-opting the AR axis, epithelial cells may become less dependent on andromedins and mesenchymal AR for androgen- regulated proliferation. The initial vulnerability of androgen-sensitive prostate cancers to hormone ablation and the eventual over-expression of the AR axis in castrate-resistant disease demonstrate the importance of AR in prostate cancer. Indeed, it has been proposed that the reliance on AR constitutes “lineage addiction”, where the growth and survival of prostate cancer cells depends on factors that regulate normal proliferation and homeostasis (Garraway and Sellers 2006).

A consequence of AR signalling in prostate cancer is the continued expression of kallikreins. This study confirmed the expression of KLK2, 3, 4, 14 and 15 by benign and malignant luminal epithelial cells, using immunohistochemistry of patient specimens as well as RT-PCR and Western blots of prostate cell lines with a luminal phenotype. A similar mRNA expression

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profile was also observed for KLK1 and KLKP1 (KRIP1). Apart from KLK14, these genes are located adjacent to one another at the centromeric end of the kallikrein locus and are all up- regulated by androgens. In comparison, KLK14 lies at the far telomeric end of the locus and is suppressed by androgens. Many previous reports have measured kallikrein levels after a single dose of hormones at one extended time point. This makes it difficult to determine whether changes are due to direct regulation by a specific hormone receptor, or secondary effects of steroid hormone metabolism, promiscuous binding to other receptors or altered cellular proliferation and differentiation. In this study, time-course, dose response, bicalutamide and AR knockdown experiments all confirmed that changes in kallikrein expression were due to direct actions of AR. Fold changes vary with experimental design, so it has not previously been possible to quantitatively compare the degree of androgen regulation of different kallikreins genes examined in separate studies. By comparing the expression of the whole kallikrein locus in two prostate cancer cell lines, this study showed that KLK2 and KLK3 are the most potently androgen-regulated genes and are surrounded by more moderately androgen-regulated kallikreins. These results demonstrates that KLK1-4 and 15 form a cluster of androgen-regulated “prostatic” kallikreins.

It will be important to characterise the co-ordinated in vivo expression profile of kallikreins in future studies. The immunohistochemistry experiments in this project show that KLK2, 3, 4, 14 and 15 expression persists in prostate cancer specimens. However, sample to sample variation in staining among the small number of specimens meant that it was not possible to accurately compare kallikrein expression in benign versus malignant glands or different stages of prostate cancer. Given that bicalutamide treatment and hormone deprivation abrogate in vitro KLK2, 3, 4 and 15 expression, it is likely that these kallikreins are also down-regulated in men during the initial stages of hormone ablation and then re-expressed when AR signalling is re-activated in castrate-resistant disease. This pattern has already been shown for KLK2 and KLK3 in tumour xenografts and patient specimens (Stanbrough et al., 2006, Wang et al., 2008b). The expression profile of kallikreins is linked to the fate of luminal epithelial cells, which undergo apoptosis upon castration and then are revived in castrate-resistant prostate cancer. This means that KLK14 may have a complex expression profile because it is modestly down-regulated by androgens and expressed in prostate cells with luminal as well as basal phenotypes. The relative expression of kallikreins in different grades of androgen-sensitive prostate cancer is more difficult to predict from their in vitro expression profiles. KLK2, KLK3, prostatic acid phosphatase and TMPRSS2 are all well characterised androgen-regulated genes in the prostate, yet KLK3 and prostatic acid phosphatase levels decrease with cancer progression, whereas KLK2 and TMPRSS2 levels increase (Abrahamsson et al., 1988, Sinha et al., 1988, Qiu et al., 1990, Sakai et al., 1991, Yang et al., 1992, Hakalahti et al., 1993, Darson et al., 1997, Darson et al., 1999, Grande et al., 2000,

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Magklara et al., 2000b, Siivola et al., 2000, Herrala et al., 2001, Lucas et al., 2008, Sterbis et al., 2008). Several studies have reported increased KLK4, 14 and 15 expression in prostate cancer compared with benign specimens (Yousef et al., 2001c, Stephan et al., 2003, Yousef et al., 2003e, Xi et al., 2004b, Dong et al., 2005a, Michael et al., 2005, Veveris-Lowe et al., 2005, Klokk et al., 2007, Rabien et al., 2008, Ramsay et al., 2008a). Some of these studies need to be replicated, because they had small sample numbers or used anti-kallikrein primary antibodies with questionable specificity. No report has compared the expression of these kallikreins to KLK2 and KLK3. Therefore, the co-ordinated in vivo expression profile of prostatic kallikreins is still unclear. It should be further investigated in a larger cohort of patients with immunohistochemistry of serial sections, or QRT-PCR of RNA extracted from laser capture microdissected tissue.

If kallikreins are found to be differentially expressed in prostate cancer progression, this would imply that their parallel in vitro response to androgens becomes uncoupled in vivo. This may occur if the expression of each kallikrein is controlled by a different combination of AR co- regulator proteins that have different expression profiles in prostate cancer. Recent ChIP-ChIP studies have shown that the DNA binding motifs of several cis-acting transcription factors are commonly located near AREs. These factors include GATA2, Oct1, FoxA1, NFI, C/EBPβ and ETS1 (Massie et al., 2007, Wang et al., 2007, Jia et al., 2008). Of note, FoxA1, NFI, and C/EBPβ knockdown have similar effects on KLK2 and KLK3 expression, whereas GATA2 knockdown decreases KLK3 but does not change KLK2 levels (Jia et al., 2008). These knockdown experiments could be extended to other prostatic kallikreins to compare the role of each co-regulator in the expression of different kallikrein genes.

The kallikrein locus is a useful model to investigate AR signalling in prostate cancer. Numerous studies have used KLK3 as a prototypical androgen-regulated gene to characterise AR binding to AREs and to explore crosstalk between AR and other signalling pathways (Kim and Coetzee 2004). However, it is unlikely that KLK3 always represents the spectrum of androgen-regulated genes. By analysing a range of prostatic kallikreins and other androgen-regulated genes, such as TMPRSS2 and NKX3.1, generic changes in AR signalling can be distinguished from gene- or locus-specific effects. Furthermore, differences in the magnitude of changes in androgen- regulated kallikrein expression could be used to estimate the potency of AR signalling. For example, a significant increase in KLK2 and KLK3, but not KLK4 or KLK15, expression would indicate more modest levels of AR activity compared with up-regulation of all four kallikreins.

The localisation of kallikreins is also altered in prostate cancer. Breakdown of the basement membrane and loss of glandular architecture in prostate cancer causes kallikreins to leak into the tumour microenvironment. Considering that kallikreins participate in enzyme cascades, the

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functional consequences of increased interstitial kallikrein concentrations depend on the repertoire of kallikreins that are expressed. For instance, this means that KLK2 may have a direct role in cancer progression by cleaving certain substrates and an indirect role by activating KLK3, which subsequently cleaves other proteins. This also implies that it is difficult to deduce the in vivo functions of individual kallikreins from the limited knowledge of their in vitro substrates. Since serine proteases regulate Nodal activity by cleaving its prodomain, the ability of kallikreins to activate pro-Nodal was investigated in this study. KLK3, which has chymotrypsin- like specificity, was unable to cleave pro-Nodal, whereas KLK4 and KLK14, which have trypsin-like specificity, cleaved pro-Nodal, but only at high enzyme concentrations. Therefore, Nodal is unlikely to be an in vivo kallikrein substrate. Given that the prostatic kallikreins are abundant in the prostate and have a co-ordinated or at least, overlapping, expression profile, future studies that identify their individual substrates and combined actions through enzyme cascades are needed. Novel proteomic techniques such as terminal amine isotopic labeling of substrates (TAILS), which identifies new N-termini that are generated by protease cleavage, would be particularly useful (Doucet and Overall 2008).

6.1.2 Exploiting the Convergence Between Prostate Cancer and Early Development In addition to the AR axis, several other signalling pathways that regulate foetal prostate development and adult tissue renewal are over-expressed in prostate cancer. Based on the morphology of patient tumours, it has long been postulated that cancer progression imitates embryonic development (Lobstein 1829, Cohnheim 1882, Bailey and Cushing 1925). More recently, large-scale gene expression techniques, such as microarrays and serial analysis of gene expression, have made it possible to make thorough comparisons between cancer and development. For example, human medulloblastoma, lung and prostate cancer specimens all have similar gene expression signatures to those observed during the development of their corresponding tissues in mice (Kho et al., 2004, Liu et al., 2006, Pritchard and Nelson 2008, Schaeffer et al., 2008). Furthermore, a group of genes that is regulated by the polycomb group protein, Bmi-1, in peripheral nervous system tissue stem cells is differentially expressed in prostate cancer, and predict poorer patient outcome (Glinsky et al., 2005). Embryonic stem cell gene expression signatures also correlate with an aggressive and poorly differentiated phenotype for breast and bladder cancer and glioblastoma (Ben-Porath et al., 2008). These studies demonstrate that cancer is a caricature of development. But what stage of development does cancer mimic? Similarities between tumour cells and embryonic stem cells, tissue anlagen and normal tissue stem cells were all noted in these studies. The developmental stage that tumour cells imitate depends on the type of cancer. Like embryos, teratocarcinomas arising from germ cells in the testes can differentiate into ectodermal, endodermal and mesodermal cell types

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(Andrews et al., 2005). In contrast, leukemias emulate leucopoiesis (Greaves et al., 1983, Greaves 1986) and prostate cancers replicate basal to luminal differentiation (van Leenders and Schalken 2003). The plasticity of the cancer depends on the plasticity of the cell of origin. Prostate cancer potentially arises from prostate epithelial stem cells or precursor cells which are committed to prostatic differentiation (Lawson and Witte 2007). Prostate cancer cells can transiently mimic mesenchymal, neuroendocrine, and bone phenotypes, but there is no evidence that prostate cancer cells can genuinely differentiate into these lineages (Koeneman et al., 1999, Lawrence et al., 2007, Yuan et al., 2007). Pierce and Speers (1988) argue that “carcinogenesis does not alter the original histiotypic determination; rather, it superimposes the malignant phenotype on it”. This suggests that at the cellular level prostate cancer more closely resembles a dysregulated form of prostate development or tissue renewal than embryogenesis.

At the molecular level, similar factors are expressed in embryos, developing tissues, adult stem cells and tumours. There is a limited repertoire of pathways that regulate stemness and these signals are interpreted in a cell-specific manner. For instance, the Wnt/β-catenin and TGFβ superfamily/Smad axes are important in embryogenesis, tissue renewal and cancer progression. The commonality in signalling molecules suggests that the embryonic stem cell gene expression signatures that are enriched in aggressive cancer cells may also apply to adult tissue stem cells (Ben-Porath et al., 2008). Unfortunately, it is difficult to make such comparisons between cancer progression and tissue renewal, because tissue stem cells and their niches are not well characterised. Nevertheless, tissue recombination experiments showing that UGM directs the prostatic differentiation of hESCs, UGE, prostate tissue stem cells and prostate cancer demonstrate that different cell types with stem cell properties can respond to the same set of regulatory cues (Cunha 1972a, Hayward et al., 1998, Taylor et al., 2006, Leong et al., 2008).

Stem cells and cancer cells are also responsive to one another. Many studies have shown that embryonic microenvironments can reprogram tumour cells to less aggressive phenotypes, presumably by restoring the balance to developmental signalling cascades that are co-opted and corrupted in cancer (DeCosse et al., 1973, Brinster 1974, Mintz and Illmensee 1975, Podesta et al., 1984, Gerschenson et al., 1986, Chung et al., 1990, Hayashi et al., 1990, Hayashi and Cunha 1991, Kulesa et al., 2006, Postovit et al., 2006, Postovit et al., 2008b). In the majority of these studies, tumour cells were exposed to foetal rudiments of their corresponding tissues. For example, breast cancer cells were co-cultured with embryonic mammary mesenchyme (DeCosse et al., 1973), melanoma cells were treated with conditioned media from embryonic skin (Gerschenson et al., 1986), and prostate cancer cells were recombined with UGM (Chung et al., 1990, Hayashi et al., 1990, Hayashi and Cunha 1991). An alternative approach using stem cells and embryos has also reduces the aggressiveness of cancer cells (Lee et al., 2005, Postovit et al.,

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2006, Postovit et al., 2008b). Each technique may cover a different spectrum of signalling pathways. Developmental mesenchyme may be particularly useful for identifying paracrine factors that regulate epithelial and mesenchymal interactions during tissue morphogenesis and are reactivated in cancer. In contrast, experiments with stem cells and embryos are likely to encompass both paracrine and autocrine signalling pathways.

In this study, prostate cancer cells were cultured on hESC CMTX. The normal regulatory cues from this embryonic microenvironment reduced the clonogencity and invasiveness of the prostate cancer cells as well as their ability to undergo vasculogenic mimicry. Greater changes were observed for DU145 compared with PC-3 cells. Even though prostate cancer more closely resembles prostate development than embryogenesis at the cellular level, the similarity between signalling cascades at the molecular level means that prostate cancer cells are also sensitive to hESC-derived factors. Collectively, this project and previous studies show that normal embryonic and developmental microenvironments reduce the aggressiveness of prostate cancer cells (Chung et al., 1990, Hayashi et al., 1990, Hayashi and Cunha 1991). Another stem cell microenvironment that could be tested is the normal prostate stem cell niche. However, the prostate stem cell niche is poorly defined, although clearly perturbed in carcinogenesis and may be difficult to study because the prostate is a slowly renewing tissue (Risbridger and Taylor 2008).

6.1.3 The Role of the Nodal Axis in Prostate Cancer The convergence between signalling pathways in development and cancer has recently been used to identify novel factors that are involved in tumour progression. For example, the observation that melanoma cells can instruct zebrafish embryos to form ectopic outgrowths and body axes led to the finding that they express the embryonic morphogen Nodal (Topczewska et al., 2006). Inhibition of Nodal signalling by hESC-derived Lefty was shown to be partially responsible for reducing the aggressiveness of melanoma cells exposed to hESC CMTX (Postovit et al., 2008b). Given that prostate cancer cells are also sensitive to hESC CMTX, the expression of Nodal in prostate cancer cells exposed to this embryonic microenvironment was examined in this project. DU145 and PC-3 cells were both shown to express Nodal which was down-regulated in DU145 cells cultured on hESC CMTX. Expression of the Nodal axis was further characterised in a range of prostate cell lines. Whereas Nodal was only expressed in DU145 and PC-3 cells, Cripto was detected in all cell lines. No Lefty A or B expression was detected using primers that amplify both transcripts. Breast cancer and melanoma cell lines also fail to express Lefty, indicating that Nodal signalling is highly dysregulated in cancer compared with embryogenesis (Postovit et al., 2008b). Using immunohistochemistry, increased Nodal

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expression was observed in malignant compared with benign glands. Therefore, the Nodal axis is a novel example of a developmental signalling cascade that is reactivated in prostate cancer.

The Nodal pathway is functionally active in prostate cancer cells. Recombinant Nodal treatments stimulated Smad2 phosphorylation in DU145 and LNCaP cells. In accordance with previous studies, high concentrations of Nodal were required to activate the Smad cascade, possibly because Cripto reduces the efficiency of the activin receptor complex (Kumar et al., 2001, Vallier et al., 2005, Kelber et al., 2008, Zhang et al., 2008). To further investigate the functional role of Nodal in prostate cancer, LNCaP cells were stably transfected with a construct encoding Flag-tagged pre-pro-Nodal. De novo expression of Pitx2, an embryonic transcriptional target of Nodal signalling, was observed in these cells. Furthermore, in soft agar assays, the clonogenicity of LNCaP cells transfected with Nodal was increased compared with empty vector control cells. Further studies could extend these results with functional assays that measure proliferation, migration and invasion and by identifying additional genes that are regulated by Nodal signalling. The potential role of Nodal in prostate development and tissue renewal is also an intriguing possibility. Based on studies of crown gall plant tumours, Braun postulated that “if a tumour makes a growth factor, that factor will be made by the cognate normal cell lineage during its development” (quoted in Pierce and Speers, 1988). Of note, some of the normal counterparts to leukemia cells are most abundant during foetal development and tissue renewal (Greaves et al., 1983, Greaves 1986). Future studies should investigate whether Nodal is expressed in developing mouse or rat prostate as well as during androgen-stimulated tissue renewal after castration-induced regression.

6.1.4 Interactions Between Androgen Receptor and Developmental Signalling Pathways Developmental pathways are expressed in embryogenesis, foetal tissue morphogenesis and adult tissue renewal, but their downstream effects are context-dependent. One way that these signals are interpreted in a cell-specific manner is through cross-regulation with other cascades. The same applies to prostate cancer. Given that the AR is essential for the growth and survival of prostate cancer cells, its association with two developmental pathways, the Wnt/β-catenin and Nodal/Smad cascades, was examined in this project. The synergistic interaction between β- catenin and the AR is well characterised (Verras and Sun 2006). Previous studies have reported that β-catenin is recruited to the proximal promoter of KLK3 (Amir et al., 2003, Li et al., 2004, Liu et al., 2008a). β-catenin has also been shown to stimulate the activity of a KLK2 reporter construct (Chesire et al., 2002). In this study, stable knockdown of β-catenin reduced the androgen-regulated expression of endogenous KLK2, 3, 4 and 15 in LNCaP cells. Initial experiments where β-catenin levels were stabilised by inhibiting GSK3β indicated that β-catenin

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may also stimulate KLK14 expression, possibly through TCF rather than AR. However, subsequent experiments demonstrated that GSK3β inhibitors up-regulate KLK14 expression independently of β-catenin. Whereas the Wnt/β-catenin cascade enhanced the expression of androgen-regulated kallikreins, Nodal antagonised AR signalling. Transfecting increasing amounts of the pre-pro-Nodal construct into LNCaP cells led to a dose responsive decrease in KLK3 reporter activity. Furthermore, there was reduced endogenous expression of androgen- regulated genes including KLK2, 3 and 15, TMPRSS2 and NKX3.1 in LNCaP cells stably transfected with Nodal. The molecular basis of these observations is likely to be the interaction between receptor-activated Smads and the AR (Hayes et al., 2001, Kang et al., 2001, Kang et al., 2002, Qiu et al., 2007). These results show that the Wnt/β-catenin and Nodal/Smad pathways have different effects on AR activity and reinforce the usefulness of the kallikrein locus as a model of androgen-regulated gene expression.

6.1.5 Final Conclusions Prostate cancer is a symptom of aging but a problem of development. Prostate cancer cells reactivate and dysregulate developmental signalling pathways associated with stemness. However, they remain committed to prostatic differentiation and reliant on the AR. As summarised in Figure 5.1, these concepts were further investigated leading to the following key findings:

1) A subset of kallikreins (KLK2, 3, 4, 14 and 15) are co-ordinately expressed in prostate luminal cells, but differentially regulated by androgens. 2) Androgen-regulated kallikrein expression is stimulated by the Wnt/β-catenin pathway, but inhibited by the Nodal/Smad cascade 3) Regulatory cues from embryonic microenvironments reduce the aggressiveness of prostate cancer cells. 4) The Nodal axis is re-expressed in prostate cancer and has a potential functional role in prostate cancer progression. Collectively, these results demonstrate that prostate cancer cells co-opt and corrupt developmental signalling cascades which have prostate-specific effects due to crosstalk with the AR.

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Figure 6.1. Androgen receptor signalling and loss of cellular differentiation are important aspects of prostate cancer progression Kallikrein expression is regulated by AR and modulated by interactions between the AR and Wnt and Nodal cascades. The reactivation of the developmental Nodal and Wnt pathways in prostate cancer is associated with loss of differentiation. Kallikrein enzyme cascades as well as the Nodal and Wnt pathways may directly facilitate the progression of prostate cancer. AR signalling prevents the complete loss of luminal differentiation by maintaining the expression of luminal-specific genes such as prostatic kallikreins. Green arrows indicate stimulatory interactions, whereas red arrows indicate inhibitory interactions. Solid arrows denote the findings of this project.

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Appendices

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Appendix A: A Summary of Studies Examining Kallikrein Expression in Prostate Cancer

Gene Results Samples (Benign:Cancer) Method References KLK2 Cancer>Benign Various Serum ELISA (Kwiatkowski et al., 1998, Partin et al., 1999, Stephan et al., 2007) Cancer>PIN>Benign 257 patients ISH (Darson et al., 1997) Metastases>Cancer>Benign 151 patients IHC (Darson et al., 1999) (primary tumours and lymph node metastases) Benign>Cancer 14 matched ELISA (Magklara et al., 2000b) Benign>Cancer 7:5 Time resolved (Siivola et al., 2000) fluorescence Cancer>Benign 27 patients ISH (Herrala et al., 2001) Cancer>Benign 6:6 IHC (Veveris-Lowe et al., 2005) KLK3 Cancer>Benign Various Serum ELISA (Kuriyama et al., 1980, Stamey et al., 1987, Lilja et al., 2008) Benign>Cancer 16:20 and 10 matched ISH, IHC (Qiu et al., 1990) Benign>Cancer 22:16 Northern, ISH, IHC (Hakalahti et al., 1993) Benign>Cancer 22:20 ELISA (Yang et al., 1992) Decrease with Cancer Progression 42 patients Radioimmunoassay (Grande et al., 2000) Cancer>Benign 24:74 ELISA (Culkin et al., 1995) Benign>Cancer 13:15 QRT-PCR (Kaushal et al., 2008) Benign>Cancer 242 matched LCM QRT-PCR (Sterbis et al., 2008) KLK4 Cancer>Benign 20 matched RT-PCR (Obiezu et al., 2002) Benign>Cancer 21 matched ELISA

Gene Results Samples (Benign:Cancer) Method References No Change 4:4 IHC (Day et al., 2002) 6:24 QRT-PCR * Cancer>Benign 518 cores ISH (Xi et al., 2004a) No Change 16:18 ELISA (Obiezu et al., 2005) Cancer>Benign 6 IHC (Veveris-Lowe et al., 2005) * Cancer>Benign 42:207 IHC (Klokk et al., 2007) Cancer>Benign 6:6 QRT-PCR (Dong et al., 2005b) 6:2 IHC Cancer>Benign 6 IHC (Ramsay et al., 2008a) KLK5 Benign>Cancer 29 matched RT-PCR (Yousef et al., 2002a) * Cancer>Benign 23 matched RT-PCR (Kurlender et al., 2004) * No Change 29 matched RT-PCR (Yousef et al., 2004b) Benign>Cancer 17:26 Serum ELISA (Sardana et al., 2007) KLK6 No Change (Below cut-off) 41:40 Serum ELISA (Diamandis et al., 2000) Benign>Cancer 17:26 Serum ELISA (Sardana et al., 2007) KLK7 Decrease in PCa 20 normal, 50 benign, 103 cancer IHC, RT-PCR (Xuan et al., 2008) KLK8 Benign>Cancer (not significant) 127:123 Serum ELISA (Parekh et al., 2007) KLK10 Benign>Cancer 7 matched RT-PCR (Yousef et al., 2005) No Change 40:41 Serum ELISA (Luo et al., 2001) No Change 127:123 Serum ELISA (Parekh et al., 2007) KLK11 Cancer>Benign 17:26 Serum ELISA (Sardana et al., 2007) Benign>Cancer 64:86 Serum ELISA (Nakamura et al., 2003b)

Gene Results Samples (Benign:Cancer) Method References Cancer>Benign 76 matched QRT-PCR (Nakamura et al., 2003c) Increase with Gleason grade 66 RT-PCR (Stavropoulou et al., 2005) Cancer>Benign 42:22 RT-PCR (Scorilas and Gregorakis 2006) KLK12 Altered localisation 82 IHC (Memari et al., 2007) KLK14 Benign>Cancer 10 matched RT-PCR (Yousef et al., 2001b) Cancer>Benign, 100 matched QRT-PCR (Yousef et al., 2003e) Cancer>Benign Not specified Serum ELISA (Borgono et al., 2007b) Cancer>Benign 28:31 Serum ELISA (Borgono et al., 2003) No Change 25 matched LCM QRT-PCR (Rabien et al., 2008) Increase with stage, relapse 186 IHC KLK15 Cancer>Benign 29 matched RT-PCR (Yousef et al., 2001c) Cancer>Benign 90 matched QRT-PCR (Stephan et al., 2003) * Cancer>Benign 12 matched RT-PCR (Michael et al., 2005) KLK31P Benign>Cancer>Metastases 10:10:5 LCM QRT-PCR (Lu et al., 2006) KRIP1 Benign>Cancer 13:15 QRT-PCR (Kaushal et al., 2008)

* Denotes studies that focussed on alternatively spliced kallikreins transcripts.

Appendix B: Steroid Hormone Regulation of Kallikrein-Related Serine Peptidases

Gene Hormone Model Methods Result References KLK1 Androgen Rat submandibular gland IHC, enzyme activity, Northern Increase (Chao and Margolius 1983, Miller et al., blot, radioimmunoassay 1984, Clements et al., 1986, Gerald et al., Oestrogen Rat pituitary, endometrium, liver IHC, enzyme activity, Northern Increase 1986, Powers 1986, Fuller et al., 1988, and kidney blot, RT-PCR Clements et al., 1990, Rosewicz et al., 1991, Chen et al., 1992, Clements et al., Progestin Rat liver Northern blot Increase 1994) Glucocorticoid ARJ42 pancreatic cells Northern blot, Western blot Decrease (Dexamethasone) KLK2 Androgen (various) LNCaP, BT-474, T-47D, PC-3 Luciferase assays, EMSA, Increase (Young et al., 1992, Murtha et al., 1993, with AR co-transfection ELISA Hsieh et al., 1997, Shan et al., 1997, Sun et Oestrogen 22Rv1, LNCaP ELISA, RT-PCR Increase al., 1997, Yu et al., 1999, Magklara et al., 2000a, Mitchell et al., 2000b, Shaw and (17β-Estradiol) T-47D Decrease Diamandis 2008) BT-474, T-47D No Change Progestin 22Rv1, LNCaP, T-47D, BT-474 ELISA, RT-PCR Increase (Norgestrel) (11-β-H-Progesterone) T-47D, PC-3 with PR ELISA, RT-PCR, Luciferase Increase assay Glucocorticoid T-47D, PC-3 with GR ELISA, RT-PCR, Luciferase Increase (Dexamethasone) assay Mineralcorticoid T-47D, BT-474 ELISA, RT-PCR Increase (Aldosterone) KLK3 Androgen (various) LNCaP DNase1 footprinting, promoter Increase (Riegman et al., 1991, Cleutjens et al., constructs, EMSA, ChIP, etc 1996, Schuur et al., 1996, Cleutjens et al.,

Gene Hormone Model Methods Result References Oestrogen 22Rv1, LNCaP ELISA, RT-PCR Increase 1997, Hsieh et al., 1997, Shan et al., 1997, Sun et al., 1997, Huang et al., 1999, (17β-Estradiol) T-47D Decrease Magklara et al., 2000a, Shang et al., 2002, BT-474 No Change Shaw and Diamandis 2008) Progestin 22Rv1, LNCaP, T-47D, BT-474 ELISA, RT-PCR Increase (Norgestrel) (11-β-H-Progesterone) T-47D, PC-3 with PR ELISA, Luciferase Assay, RT- Increase PCR Glucocorticoid T-47D, PC-3 with GR ELISA, Luciferase Assay, RT- Increase (Dexamethasone) PCR BT-474 ELISA, RT-PCR No Change Mineralcorticoid T-47D, BT-474 ELISA, RT-PCR Increase (Aldosterone) KLK4 Androgen LNCaP, BT-474, CWR22 Northern blot, Western blot, Increase (Nelson et al., 1999b, Yousef et al., 1999b, (Testosterone, DHT, xenograft QPCR, RT-PCR Dong et al., 2001, Korkmaz et al., 2001, R1881) Myers and Clements 2001, Xi et al., 2004b, Dong et al., 2005b, Lazarevic et al., Oestrogen LNCaP, KLE, OVCAR-3 Northern blot, Western blot, RT- Increase 2008, Lai et al., 2009) (17β-Estradiol) PCR BT-474 RT-PCR No Change Progestin LNCaP, T47-D Northern blot, Western blot, RT- Increase (11-β-H-Progesterone) PCR, QPCR, promoter constructs, ChIP (Norgestrel) BT-474 RT-PCR Increase Glucocorticoid LNCaP Northern blot Increase (Dexamethasone)

Gene Hormone Model Methods Result References

KLK5 Androgen (DHT) Ms-751, PC3(AR)6, BT-474 RT-PCR, ELISA Increase (Yousef et al., 2003b, Yousef et al., 2004b, Jeong et al., 2005, Paliouras and Diamandis 2007, Shaw and Diamandis Oestrogen MCF-7, HTB-75 PC3(AR)6, BT- ELISA, RT-PCR No Change 2008) (17β-Estradiol) 474 MCF-10A, HaCat, Caski, MCF-7, Increase BT-474 Progestin MCF-7, HTB-75, BT-474 ELISA, RT-PCR No Change

(Norgestrel) PC3(AR)6, Ms-751 Increase PR KO mouse uteri Microarray, QPCR Increase (WT vs KO)

Glucocorticoid MCF-7, PC3(AR)6 ELISA No Change (Dexamethasone) HTB-75, MCF10A, Ms-751, Me- Decrease 180, Ht-3, Caski HTB-75, HaCat, MDA-MB-468 Increase Mineralcorticoid MCF-7, T-47-D, HTB-75 ELISA, RT-PCR No Change (Aldosterone) PC3(AR)6, BT-474 KLK6 Androgen (DHT) BT-474 RT-PCR Increase (Yousef et al., 1999a, Jeong et al., 2005, BG-1, HTB-75, OvCAR3 ELISA No Change Paliouras and Diamandis 2007, Shan et al., 2007, Shaw and Diamandis 2008) Oestrogen BT-474, HaCat, Caski, MCF-7, T- RT-PCR, ELISA Increase (17β-Estradiol) 47D BG-1, HTB-75, OvCAR3 No Change Progestin BT-474 RT-PCR Increase (Norgestrel) BG-1, HTB-75, OvCAR3 ELISA No Change PR KO mouse uteri Microarray, QPCR Increase (WT vs KO)

Gene Hormone Model Methods Result References Glucocorticoid BG-1, HTB-75, OvCAR3, BT-474, ELISA No Change (Dexamethasone) T-47D MCF-7 Increase Ms-751, Ht-3, Caski Decrease Mineralcorticoid T-47D, BT-474, MCF-7 ELISA, RT-PCR No Change (Aldosterone) KLK7 Oestrogen BT-474 RT-PCR Increase (Yousef et al., 2000e, Shaw and Diamandis (Unknown ligand) 2008) Glucocorticoid BT-474 RT-PCR Increase (Unknown ligand) (Dexamethasone) HaCat ELISA Increase

KLK8 Androgen (DHT) T47D, MCF-7, PC3(AR)6 ELISA, RT-PCR Increase (Katsu et al., 2002, Kishi et al., 2006, Oestrogen MCF-7, T47D ELISA Increase Paliouras and Diamandis 2007, Shaw and Diamandis 2008) (17β-Estradiol) PC3(AR)6 No Change (Diethylstilbestrol) Mouse vaginal epithelium Northern blot Increase

Progestin T-47D, MCF-7, PC3(AR)6 ELISA Increase (Norgestrel) Glucocorticoid T-47D, MDA-MB-435, MCF-7 ELISA, RT-PCR Increase (Dexamethasone) Ms-751, Ht-3, Caski Decrease

Mineralcorticoid T-47D, MCF-7, PC3(AR)6 ELISA, RT-PCR No Change (Aldosterone) KLK9 Androgen (DHT) BT-474 RT-PCR Increase (Yousef and Diamandis 2000, Yousef et Oestrogen BT-474, HTB-75, MCF-7, T-47D RT-PCR, ELISA, QPCR Increase al., 2001a, Shaw and Diamandis 2008) (17β-Estradiol)

Gene Hormone Model Methods Result References Progestin BT-474, MCF-7, T47-D RT-PCR, QPCR Increase (Norgestrel) KLK10 Androgen (DHT) T-47D, MCF-7, BT-474 ELISA, RT-PCR Increase (Luo et al., 2000, Luo et al., 2001, MDA-MB-468, MDA-MB-468, No Change Kulasingam and Diamandis 2007, MCF7 Paliouras and Diamandis 2007, Paliouras and Diamandis 2008, Shaw and Diamandis Oestrogen BT-474, MCF-7 ELISA, RT-PCR Increase 2008) (17β-Estradiol) T-47D, MCF-10A, MDA-MB-468 No Change Progestin MCF-7, BT-474 ELISA, RT-PCR Increase (Norgestrel) T-47D, MCF-10A No Change Glucocorticoid MCF-7, MDA-MB-468, Ht-3 ELISA, RT-PCR Increase (Dexamethasone) T-47D No Change MCF-10A Decrease (Betamethasone) MDA-MB-468, T-47D Increase MCF-10A Decrease KLK11 Androgen (DHT) BT-474, MCF-7, Me-180, T-47D ELISA, RT-PCR Increase (Yousef et al., 2000d, Paliouras and Oestrogen BT-474, MCF-7, Me-180 ELISA, RT-PCR Increase Diamandis 2007, Paliouras and Diamandis 2008, Shaw and Diamandis 2008) (17β-Estradiol) Progestin MCF-7 ELISA, RT-PCR Increase (Norgestrel) Glucocorticoid MCF-7 ELISA, RT-PCR Increase (Dexamethasone) Me-180, Ht-3 No Change KLK12 Androgen (DHT) BT-474, LNCaP, T-47D RT-PCR Increase (Yousef et al., 2000c) (Mibolerone) BT-474, LNCaP, T-47D Increase

Gene Hormone Model Methods Result References Oestrogen BT-474 RT-PCR Increase (17β-Estradiol) LNCaP, T-47D No Change Progestin (Norgestrel) BT-474, LNCaP, T-47D RT-PCR Increase Glucocorticoid BT-474 RT-PCR Increase (Dexamethasone) T-47D No Change LNCaP Decrease Mineralcorticoid BT-474 RT-PCR Increase (Aldosterone) LNCaP, T-47D No Change KLK13 Androgen (DHT) BT-474, T-47D RT-PCR, ELISA Increase (Yousef et al., 2000a, Paliouras and Oestrogen BT-474, MCF-7 RT-PCR Increase Diamandis 2007, Paliouras and Diamandis 2008, Shaw and Diamandis 2008) (17β-Estradiol) Progestin (Norgestrel) BT-474 RT-PCR Increase Glucocorticoid T-47D, Ht-3 RT-PCR, ELISA Increase (Dexamethasone) KLK14 Androgen (DHT) BT-474, T-47D, ZR-75, BG-1, RT-PCR Increase (Borgono et al., 2003, Yousef et al., 2003a, HTB-75 Paliouras and Diamandis 2007, Paliouras Oestrogen BT-474 RT-PCR, ELISA Increase and Diamandis 2008) (17β-Estradiol) BT-474, T-47D, ZR-75, BG-1, No Change HTB-75 Progestin (Norgestrel) T-47D, ZR-75, BT-474, MCF-7 RT-PCR, ELISA Increase T-47D, BG-1, BT-474, HTB-75 No Change Glucocorticoid T-47D, BT-474, MCF7 RT-PCR, ELISA No Change (Dexamethasone) Mineralcorticoid BT-474 RT-PCR No Change (Aldosterone)

Gene Hormone Model Methods Result References KLK15 Androgen (DHT) BT-474, LNCaP RT-PCR Increase (Yousef et al., 2001c, Yousef et al., 2002b, MCF-7 No Change Shaw and Diamandis 2008) Oestrogen BT-474, LNCaP RT-PCR Increase (17β-Estradiol) MCF7 No Change Ov-90 ELISA Decrease Progestin (Norgestrel) LNCaP RT-PCR Increase BT-474, MCF7 No Change Glucocorticoid LNCaP RT-PCR Increase (Dexamethasone) Mineralcorticoid LNCaP RT-PCR Increase (Aldosterone) KLKP Androgen (R1881) LNCaP QPCR Increase (Lu et al., 2006, Kaushal et al., 2008) 1

Appendix C: Oligonucleotide Primer Sequences

Gene Primer Sequences (5’ to 3’) Reference/Source 18S Ribosomal RNA F – GATCCATTGGAGGGCAAGTCT R - CCAAGATCCAACTACGAGCTTTTT (Lau and Kolodner 2003) β-catenin F – CGTGCAATCCCTGAACTGACA R - TGAGGAGAACGCATGATAGCG Androgen Receptor F – CTGGACACGACAACAACCAG R - CAGATCAGGGGCGAAGTAGA (Kaushal et al., 2008) (QRT-PCR only) Androgen Receptor F - AGCCCCACTGAGGAGACAACC R - ATCAGGGGCGAAGTAGAGCATC (Pitkanen-Arsiola et al., (RT-PCR only) 2006) E-cadherin F - GCCCATTTCCTAAAAACCTGG R - TTGGATGACACAGCGTGAGAG Cytokeratin 5 F - CAGCGTCAAATTTGTCTCCAC R - TTGGTCTAGACTACTCTCCAG (Kinouchi et al., 2002) Cytokeratin 8 F - CTGGGATGCAGAACATGAGTATTC R - GTAGCTGAGGCCGGGGCTTGT Dr Dimitri Odorico (QUT) Cytokeratin 14 F - CGCCAAATCCGCACCAAGGTC R - GAAGCAGGGTCCAGCTGTGAA (Kinouchi et al., 2002) Cytokeratin 18 F - GAGACGTACAGTCCAGTCCTTGG R - CCACCTCCCTCAGGCTGTT Dr Dimitri Odorico (QUT) Cripto F - AGAGATGACAGCATTTGGCCC R - AAAAGGACCCCAGCATGCA Furin F - GCAACACCTGGTGGTACAGA R - TCTGCGGAGTAGTCATGTGG (Lapierre et al., 2007) GAPDH F - GCAAATTCCATGGCACCGT R - TCGCCCCACTTGATTTTGG HPRT1 F - TGAACGTCTTGCTCGAGATGTG R - CCAGCAGGTCAGCAAAGAATTT Kallikrein 1 F - CGGCTCTGTACCATTTCAGCA R - CGTCGTCAAACAAGTTGTGGC Kallikrein 2 F - CTGCCCATTGCCTAAAGAAGAA R - GGCTTTGATGCTTCAGAAGGCT Kallikrein 3 F - AGTGCGAGAAGCATTCCCAAC R - CCAGCAAGATCACGCTTTTGTT (Dong et al., 2005a) Kallikrein 4 (exon 1-2) F - TACCTCATCCTTGGTGTCGCA R - ACGCCCGAGCAGAACAATT (Dong et al., 2005a) Kallikrein 4 (exon 2-3) F - GGCACTGGTCATGGAAAACGA R - TCAAGACTGTGCAGGCCCAGC (Dong et al., 2005a) Kallikrein 5 F - TGATGTTTCCTGTGACCACCC R - TGTGCATATCGCAGTCGGATC

Gene Primer Sequences (5’ to 3’) Reference/Source Kallikrein 6 F - AATAAGTTGGTGCATGGCGG R - AACTCTCCCTTTGCCGAAGGT Kallikrein 7 F - ATGGCAAGATCCCTTCTCCTG R - GGCGCCATCAATAATCTTGTC Kallikrein 8 F - GCTGCCCACTGTAAAAAACCG R - CACATCGCTGCTGTTGTAGCA Kallikrein 9 F - TCCACCTTACTCGGCTCTTCTG R - GAAGAAGTCCGTAACCCGGAAC Kallikrein 10 F - TGATCACCTGCTGCTTCTTCAG R - GGCCAGCTTTAGCAACATGAG Kallikrein 11 F - CAAGCCCCGCTACATAGTTCA R - TTGCGGTGGTCTTTGTTGG Kallikrein 12 F - CTTTGGAAGTGACCCACCATG R - TGTGAGTTACGCCCACACTCA Kallikrein 13 F - TCCCAGGAGTCTTCCAAGGTTC R - AGTAGCCGCCCTTGCACTAGTA Kallikrein 14 F - CAGCCCCTAAAATGTTCCTCC R - TGCACGTATGGCCACCAAT Kallikrein 15 F - GTGAAAGGATGGAGCTGGATG R - GGCACAATGCAACGTATCTGG KRIP1 F - TTCGGCAACTTCCAGTGCAA R - CGGAGAACTATGGTGCTGGCTA (Kaushal et al., 2008) Lefty A/B F - CTGCCGCCAGGAGATGTAC R - ACACTCATAAGCCAGGAAGCC N-cadherin F - TGCTACTTTCCTTGCTTC R - TTCTCCTCCACCTTCTTC NKX3.1 F - AACCATTTCACCCAGACAGCCT R - TGTGACAGATTGGAGCAGGGTT (Mostaghel et al., 2007) Neuron-specific enolase F - TCCTTCCCGATACATCACTGG R - CCAATCATCCTGGTCAAATGG p63 F - AGCGTTTCGTAGAAACCCCA R - CCCAGATGTGCTGGAAAACCTC PACE4 F - GTACCTCAACTTGGGCCAGA R- TCGTAGCTGGCGTAGGAATC (Lapierre et al., 2007) Pitx2 F - ACTTTACCAGCCAGCAGCTC R - CGCTCCCTCTTTCTCATTT Prostate stem cell antigen F - CCACCAGTACCATGAAGGC R - CAGTCCTCGTTGCTCACCTG Dr Dimitri Odorico (QUT) TMPRSS2 F - CCATTTGCAGGATCCGTCTG R - GGATGTGTCTTGGGGAGCAA (Mostaghel et al., 2007) Vimentin F - AACACCCTGCAATCTTTC R - CCATTTCCTCCTTCATATTC

Appendix D: Primary Antibodies Used for Western Blots and Immunohistochemistry

Target* Antibody IHC WB Source/Reference Concentration Concentration Actin Monoclonal mouse anti-actin 200 ng/mL Chemicon, Temecula, CA, USA AR Polyclonal rabbit anti-androgen receptor - 100 ng/mL Santa Cruz Biotechnology, Applied Medical, Brisbane, QLD, Australia β-catenin Monoclonal mouse anti-β-catenin - 250 ng/mL BD Biosciences Cripto Monoclonal mouse anti-cripto - 1 μg/mL R&D Systems, Bio-Scientific, Gymea, NSW, Australia E-cadherin Monoclonal mouse anti-E-cadherin ectodomain - 400 ng/mL Invitrogen FLAG Polyclonal rabbit anti-FLAG epitope - 80 ng/mL Sigma GAPDH Polyclonal rabbit anti-GAPDH - 200 ng/mL Abcam, Sapphire Biosciences, Redfern, NSW, Australia KLK2A Polyclonal rabbit anti-KLK2 catalytic domain 1 μg/mL 1 μg/mL Abcam KLK2B Polyclonal goat anti-KLK2 200 ng/mL - Santa Cruz Biotechnology KLK3A Polyclonal rabbit anti-KLK3 600 ng/mL 600 ng/mL Dako KLK3B Monoclonal mouse anti-KLK3 200 ng/mL - Neomarkers, DKSH, Hallam, VIC, Australia KLK3C Polyclonal rabbit anti-KLK3 catalytic domain 1000 ng/mL - Abcam KLK3D Monoclonal mouse anti-KLK3 125 μg/mL - R&D Systems KLK4A 1:1 mixture of polyclonal rabbit anti-KLK4 N- 1:300 dilution of - (Harvey et al., 2003) terminal and anti-KLK4 mid-region each (conc unknown) KLK4B Polyclonal rabbit anti-KLK4 kallikrein loop domain 2 μg/mL - Abcam KLK4C Polyclonal rabbit anti-KLK4 catalytic domain 2 μg/mL 2 μg/mL Abcam KLK4D Monoclonal mouse anti-KLK4 125 μg/mL - R&D Systems

Target* Antibody IHC WB Source/Reference Concentration Concentration KLK14A Polyclonal rabbit anti-KLK14 kallikrein loop domain 2 μg/mL - Abcam KLK14B Polyclonal rabbit anti-KLK14 catalytic domain 2 μg/mL - Abcam KLK14C Monoclonal mouse anti-KLK14 125 μg/mL - R&D Systems KLK14D Polyclonal rabbit anti-anti-KLK14 400 ng/mL - Abcam KLK14E Polyclonal goat anti-KLK14 400 ng/mL - Santa Cruz Biotechnology KLK15A Polyclonal rabbit anti-KLK4 kallikrein loop domain 2 μg/mL - Abcam KLK15B Rabbit anti-KLK15 catalytic domain polyclonal 2 μg/mL - Abcam antibody KLK15C Monoclonal mouse anti-KLK15 125 μg/mL - R&D Systems Laminin 5 γ2 Monoclonal mouse anti-anti-laminin 5 γ2 chain - 1 μg/mL Chemicon Nodal Polyclonal rabbit anti-human Nodal (H-110) - 400 ng/mL Santa Cruz Biotechnology Nodal Polyclonal goat anti-mouse Nodal antibody 2 μg/mL 1 μg/mL R&D Systems NSE Monoclonal mouse anti-neuron-specific enolase - 60 μg/mL Novus Biologicals, Sapphire Bioscience, Redfern, NSW, Australia Smad2/3 Polyclonal rabbit anti-Smad2/3 - 1:1000 dilution Cell Signalling Technology, Danvers, MA, (conc unknown) USA Smad2 Polyclonal rabbit anti-phosphorylated Smad2 - 1:1000 dilution Cell Signalling Technology (conc unknown) Tubulin Monoclonal mouse anti-pan-tubulin 200 ng/mL - Sigma V5 Polyclonal rabbit anti-V5 epitope - 50 ng/mL Invitrogen * To help differentiate between kallikrein antibodies they have been arbitrarily named KLK2A, KLK2B etc.

Appendix E: Expression of Kallikreins in Human Embryonic Stem Cells

KLK1-15 expression in H9 hESCs was examined using RT-PCR with 35 cycles. The house-keeping gene, HPRT1, was used as a positive control. All primers are listed in Appendix C.

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