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The kisspeptin and GPR54 ligand-receptor pair in autocrine and endocrine signalling in cancer Prentice, Leah Marie 2009

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THE KISSPEPTIN AND GPR54 LIGAND-RECEPTOR PAIR IN AUTOCRINE AND ENDOCRINE SIGNALLING IN CANCER by Leah Marie Prentice B.M.L.Sc., University of British Columbia, 2004  A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF  DOCTOR OF PHILOSOPHY  in The Faculty of Graduate Studies (Pathology and Laboratory Medicine)  THE UNIVERSITY OF BRITISH COLUMBIA (Vancouver) June 2009   Leah Marie Prentice, 2009 ii ABSTRACT Kisspeptins and their receptor, GPR54, mediate sex hormone release through stimulation of the hypothalamic-pituitary-gonadal axis and have been implicated as metastasis suppressors. Expression of kisspeptin and GPR54 has been associated with less invasive cancers as determined by RNA expression, and a multitude of in vitro studies has consistently shown that overexpression of either ligand or receptor in malignant cell lines results in a less invasive phenotype. We hypothesized that expression of GPR54/kisspeptin in epithelial malignancies is predictive of disease outcome and altering endogenous GPR54 signalling in malignant breast and ovarian epithelial cells could alter their metastatic properties. We have determined by immunohistochemistry that kisspeptin and GPR54 are independent favourable prognostic markers for ovarian carcinoma and are specific for the clear cell cancer subtype; the least characterized of the subtypes. Additionally, loss of GPR54 is associated with poor prognosis in node positive breast cancer patients and is also lost in prostate cancer and testicular germ cell nonseminomas as compared to more benign disease. Moreover, secreted kisspeptin is elevated above physiological levels in the plasma of women with gynaecological cancers, including ovarian cancer. We evaluated GPR54 expression across a panel of breast and ovarian cancer cell lines to create an in vitro model system with which to knockdown GPR54 expression using RNA interference. However, we discovered that endogenous GPR54 was internalized rather than localized to the plasma iii membrane of these cancer cell lines. Consequently, internal GPR54 was unable to signal through its canonical Gq pathway. To discover novel genes involved in kisspeptin-GPR54 signalling, we assessed gene expression differences between the Gpr54 and Kiss1 knockout mice as compared to wildtype mice. Our novel candidate list provides insight into physiological signalling in the hypothalamus that can then be applied to epithelial anti-metastatic signalling. Our results also support the sex hormone negative feedback effect on kisspeptin expression as reported in the current literature. In summary, we have confirmed kisspeptin and GPR54 as favourable prognostic markers, are the first to report the intracellular localization of GPR54 in endogenously expressing cancer cell lines, and we have introduced a list of novel genes involved in signalling.   iv TABLE OF CONTENTS Abstract ...............................................................................................................ii Table of Contents...............................................................................................iv List of Tables......................................................................................................vi List of Figures ..................................................................................................viii List of Abbreviations ..........................................................................................x Acknowledgements .........................................................................................xiii Dedication.........................................................................................................xiv Co-authorship Statement .................................................................................xv 1 Literature Review, Hypotheses and Specific Aims.................................1 1.1 Epithelial Kisspeptin...............................................................................1 1.2 Neuroendocrine Kisspeptin..................................................................15 1.3 Hypotheses and Specific Aims.............................................................31 1.4 References...........................................................................................34 2 GPR54 and KiSS1 Expression in Clinical Cases: Assessment of Prostate, Testicular, Breast, and Ovarian Cancer Tissue Microarrays.....46 2.1 Introduction ..........................................................................................47 2.2 Material and Methods ..........................................................................48 2.3 Results.................................................................................................59 2.4 Conclusions .........................................................................................70 2.5 References...........................................................................................75 3 Kisspeptin and GPR54 Immunoreactivity in a Cohort of 518 Patients Defines Favourable Prognosis and Clear Cell Subtype in Ovarian Carcinoma......................................................................................................79 3.1 Introduction ..........................................................................................80 3.2 Material and Methods ..........................................................................82 3.3 Results.................................................................................................88 3.4 Conclusions .........................................................................................98 3.5 References.........................................................................................104  v 4 Plasma Kisspeptin is Elevated in Gynaecological Malignancy .........110 4.1 Introduction ........................................................................................111 4.2 Material and Methods ........................................................................112 4.3 Results...............................................................................................119 4.4 Conclusions .......................................................................................127 4.5 References.........................................................................................130 5 Endogenous GPR54 in Cancer Cell Lines is Intracellular and Does Not Signal Through the Canonical Gq Pathway............................................135 5.1 Introduction ........................................................................................136 5.2 Material and Methods ........................................................................137 5.3 Results...............................................................................................150 5.4 Conclusions .......................................................................................169 5.5 References.........................................................................................175 6 Differential Transcription in the Hypothalamus of Gpr54 and Kiss1 Knockout Mice Revealed by One Million Exon Array Analysis ...............179 6.1 Introduction ........................................................................................180 6.2 Material and Methods ........................................................................181 6.3 Results...............................................................................................192 6.4 Conclusions .......................................................................................204 6.5 References.........................................................................................211 7 Summary, Overall Conclusions, and Future Directions .....................216 7.1 Summary............................................................................................216 7.2 Significance and Potential Applications .............................................223 7.3 Overall Conclusions ...........................................................................227 7.4 Future Directions................................................................................228 7.5 References.........................................................................................232  vi LIST OF TABLES Table 1.1 Summary of statistically significant KiSS1 and GPR54  expression in clinical cases..........................................................................14 Table 2.1 Clinicopathological characteristics of the breast TMA.........................50 Table 2.2 Clinicopathological characteristics of the prostate TMA......................51 Table 2.3 Clinicopathological characteristics of the ovarian TMA .......................53 Table 2.4 Multivariable analysis in node positive breast cancer patients ............63 Table 2.5 Kp IHC correlation with clinicopathological markers............................63 Table 2.6 GPR54 IHC correlations with clinicopathological markers ..................64 Table 2.7 GPR54 gene ratio and expression in ovarian cancer ..........................68 Table 2.8 KiSS1 gene ratio and expression in ovarian cancer............................69 Table 3.1 Clinicopathological characteristics of the cohort..................................83 Table 3.2 Multivariable disease specific proportional hazards ............................95 Table 3.3 Multivariable overall proportional hazards...........................................96 Table 3.4 Percentage of kisspeptin and GPR54 positive cases within the  histological subtypes....................................................................................99 Table 3.5 Kisspeptin and GPR54 proportions within the cohort ..........................99 Table 4.1 Characteristics of the New York clear cell array................................114 Table 4.2 Characteristics of the Edmonton clear cell array...............................114 Table 4.3 Characteristics of the gynaecological tissue microarray....................115 Table 4.4 Percentage of kisspeptin and GPR54 positive cases within the  histological subtypes..................................................................................121 Table 4.5 Kisspeptin and GPR54 proportions within the cohort ........................121 Table 4.6 Characteristics of plasma samples by group.....................................124 vii Table 5.1 Summary of antibodies used.............................................................149 Table 5.2 Summary of transfection conditions ..................................................149 Table 6.1 Gene list for the 96 Low Density Array..............................................189 Table 6.2 Gene list for the 48 Low Density Array..............................................190 Table 6.3 Genes with significant fold change from the 95 LDA assay ..............196 Table 6.4 Characteristics of hormonally treated mice .......................................198 Table 6.5 KKO hormone only effect genes compared with  testosterone treatment...............................................................................198 Table 6.6 GKO hormone only effect genes compared with  testosterone treatment...............................................................................198 Table 6.7 KKO genotype effect only genes compared with WT........................199 Table 6.8 GKO genotype effect only genes compared with WT........................199 Table 6.9 KKO genes with independent genotype and hormone effect ............200 Table 6.10 GKO genes with independent genotype and hormone effect..........200 Table 6.11 KKO hormone and genotype effect interacting genes.....................200 Table 6.12 GKO hormone and genotype effect interacting genes ....................201  viii LIST OF FIGURES Figure 1.1 The kisspeptins....................................................................................3 Figure 1.2 GPR54 intracellular signalling through the Gq pathway .....................6 Figure 1.3 Kp and GPR54 hormonal feedback regulation in the rodent..............22 Figure 1.4 Summary of human GPR54 mutations and their effects ....................28 Figure 2.1 GPR54 and Kp expression in male cancers ......................................60 Figure 2.2 Kp and GPR54 expression in breast cancer ......................................62 Figure 2.3 GPR54 and KiSS1 gene copy number in breast cancer ....................66 Figure 3.1 Western blot demonstrating GPR54 specificity..................................86 Figure 3.2 Immunohistochemistry controls..........................................................89 Figure 3.3 Immunoreactivity patterns..................................................................91 Figure 3.4 Disease specific and overall survival curves for Kp and  GPR54.........................................................................................................93 Figure 3.5 GPR54 is a favourable prognostic marker in the clear  cell subtype................................................................................................100 Figure 4.1 Disease specific survival curves for Kp and GPR54 in  ovarian cancer clear cell subtype...............................................................120 Figure 4.2 Plasma Kp correlation with malignant potential and subtype ...........125 Figure 4.3 Plasma Kp compared to IHC expression .........................................126 Figure 5.1 Comparison of GPR54 and KiSS1 expression across  cell lines .....................................................................................................151 Figure 5.2 GPR54 does not signal through Gq or Gs in mammary  cell lines .....................................................................................................153 Figure 5.3 MAPK activation by Kp stimulation of GPR54..................................156 Figure 5.4 Stable knockdown of GPR54 in TOV21G but not OV90  ovarian cells...............................................................................................160 Figure 5.5 Cell line expression of GPR54 on the plasma membrane  as determined by flow cytometry ...............................................................163 ix Figure 5.6 GPR54 is intracellular and co-localizes with  -arrestin in  TOV21G cells ............................................................................................165 Figure 5.7 Localization of GPR54 is not altered by knockdown of  endogenous Kp..........................................................................................167 Figure 6.1 Summary of the experimental procedure .........................................184 Figure 6.2 Summary of statistical grouping of the hormonally  controlled LDA assay.................................................................................191 Figure 6.3 Volcano plots from the Affymetrix Exon 1.0 ST Array ......................194 Figure 6.4 Summary of Affymetrix results .........................................................195 Figure 6.5 Summary of gene expression differences with a fold  change 1.5 ...............................................................................................202  x LIST OF ABBREVIATIONS Ab Antibody ABI Applied Biosystems ANOVA Analysis of variation Ar Androgen receptor ATP Adenine triphosphate BACs Bacterial artificial chromosome BCA Bicinchoninic acid BPH Benign prostatic hyperplasia BSA Bovine serum albumin cAMP Cyclic adenine monophosphate cDNA Complementary DNA CHO Chinese hamster ovary CI Confidence interval CK Cytokeratin Cy-5 Cyanines-5 DAG Diacyl glycerol DAPI 4,6-diamidino-2-phenylindole DDT Dithiothreitol DMEM Dulbelco's modified Eagle's medium DMSO Dimethyl sulfoxide dNTP Deoxy ribonucleotide triphosphate DSS Disease specific survival ECL Enhanced chemiluminescence EDTA Ethylenediaminetetraacetic acid EGF Epidermal growth factor EGTA Ethylene glycol tetraacetic acid EIA Enzyme immunoassay ER Estrogen receptor Erbb2 Epidermal growth factor receptor 2 xi ERK Extracellular signal-regulated kinase FBS Fetal bovine serum FISH Fluorescent in situ hybridization FITC Fluorescein isothiocyanate FSH Follicular stimulating hormone GDP Guanine diphosphate GFP Green fluorescent protein GnRH Gonadotropin releasing hormone GPR G-protein coupled receptor (GPCR) GPR54 G-protein coupled receptor 54/ hOT7T175/ AXOR12/KiSS1R GTN Gestational trophoblastic neoplasia GTP Guanine triphosphate H&E Hematoxylin and eosin hCG Human choriogonadotropin HCl Hydrochloric acid HF Hank's balanced buffer with 2% FBS HRP Horseradish peroxidase IBMX Isobutylmethylxanthine IF Immunofluorescence IHC Immunohistochemistry IOSE Immortalized ovarian surface epithelial IP3 Inositol 1,4,5-triphosphate IR Immunoreactivity/Infared Kp Kisspeptin/metastin/KiSS1 L2K Lipofectamine 2000 LDH Lactate dehydrogenase LH Lutenizing hormone MAPK Mitogen activated protein kinase min Minute MMP  Matrix metalloproteinase NaDOC Sodium deoxycholate xii NMDA N-methyl-D-aspartate acid OS Overall survival PBS Phosphate buffered saline PCR Polymerase chain reaction PI Propidium iodide PIP2 Phoshpatidylinositol 4,5-bisphosphate PKC Protein kinase C PLC Phoshpolipase C PR Progesterone receptor PVDF Polyvinylidene fluoride QPCR Quantitative/real-time polymerase chain reaction RFU Relative fluorescence units RIA Radioimmunoassay RIPA Radioimmunoprecipitation assay RT Room temperature SDS Sodium dodecyl sulphate shRNA Short hairpin siRNA Short interfering RNA TBS Tris buffered saline TBST Tris buffered saline with Tween TMA Tissue microarray WB Western blot WHO World Health Organization  xiii ACKNOWLEDGEMENTS I have been extremely fortunate to have such a wonderful group of peers and mentors to guide me through my graduate studies. I feel truly privileged that Drs. David Huntsman and Samuel Aparicio were my supervisors; they were brilliant at providing sound guidance, were always eager to discuss exciting projects, and their dedication as scientists continues to inspire me. My supervisory committee was remarkable in that all members made themselves available for individual consult on all my projects, Dr. Sandra Dunn, Dr. Michael Cox and Dr. David Walker generously provided their expertise and counsel. Drs. Blake Gilks and Tosten Neilsen also willingly addressed my inquiries and provided valuable insight. Dr. Lindsay Brown was an exceptional peer to learn from and I continue to appreciate the advice that she has provided throughout my studies and beyond. I have had the benefit of having access to a great team of scientists, students, and technicians available at; the Genetic Pathology Evaluation Centre, Jack Bell Research Centre, Centre for Translational and Applied Genomics, and the British Columbia Cancer Research Centre. I am sincerely thankful to Angela Burleigh, Melinda Carrier, Sohrab Shah, Maggie Cheang, Samuel Leung, Steve Kalloger, Janine Senz, Armelle Troussard, Dawn Cochrane, Jefferson Terry, Steven McKinney, Elizabeth Hajen, Martin Köbel Jenny Cromarty, Teresa Ruiz de Algara, Darren Saunders, Damian Yap, John Fee, Adrian Wan, Niki Boyd, Lee Smith, Erika Mehl, and Challayne Smith. xiv DEDICATION To my family,  You have all encouraged me to succeed, and with your love and support I have achieved above and beyond.     In memoriam: My mother, Sharon Diane Prentice, 1949-2002 Love you and miss you everyday  xv CO-AUTHORSHIP STATEMENT  All tissue microarrays described were already pre-existing and the original publications are referenced.  Two pathologists performed all the immunohistochemical analysis: Dr. Ashish Rajput for Chapter 3 and Dr. Martin Köbel for Chapters 2 and 4, both from the Genetic Patholgy Evaluation Centre, Vancouver, BC.  Erika Mehl, a technician from the Genetic Pathology Evaluation Centre, Vancouver, BC, ran the automated immunohistochemical staining system.  Dr. Kevin Murphy at Imperial College in London, UK, performed the radioimmunoassay to measure kisspeptin in human plasma.  The murine studies and dissections described in Chapter 6 were performed by Dr. Xavier d’Anglemont de Tassigny from the University of Cambridge, Cambridge, UK.  All statistical analysis were performed by statisticians; Steven Kalloger for Chapters 3 and 4, Dr. Steven McKinney for Chapters 2, 3, and 6, and Ozge Goktepe for Chapter 2, all from the Molecular Oncology and Breast Cancer Program, British Columbia Cancer Research Centre, Vancouver, BC. Additional analysis was performed by Samuel Leung from the Genetic Pathology Evaluation Centre, for the breast cancer tissue microarray described in Chapter 2.  xvi The introduction and conclusion for Chapter 3 were co-written with Dr. Samuel Aparicio.  All study designs were collaborations between the candidate, Dr. David Huntsman, and Dr. Samuel Aparicio. Chapter 3 also had Dr. Peter Leung contribute to the study design and Chapter 6 had Dr. William College as a study design contributor.  Excluding what has been noted above, the candidate performed all the experiments, wrote all drafts of the manuscripts, performed all revisions of the manuscripts, and created all the figures and tables for the manuscripts. The candidate assisted and conferred with the pathologists, technician and statisticians for all the immunohistochemical projects as well as conferred with Steven McKinney for the Affymetrix and real-time PCR analysis presented in Chapter 6.  Drs. David Huntsman and Samuel Aparicio were instrumental in the critique of this dissertation.   1 1 LITERATURE REVIEW, HYPOTHESES AND SPECIFIC AIMS  Since the discovery of the anti-metastatic KiSS1 gene and its encoded kisspeptins over 10 years ago, there has been a flurry of publications attempting to describe the mechanism responsible for the anti-invasive phenotype associated with kisspeptin overexpression. The kisspeptin story became more complex upon the discovery of its G-protein coupled receptor GPR54 being mutated in idiopathic hypogonadotropic hypogonadism. Subsequent studies demonstrated the ligand-receptor pair as key players in regulating the hypothalamic-pituitary-gonadal axis in many species including humans. The focus of my research has been to assess if kisspeptin and GPR54 as anti- metastatic agents could be used as predictive markers for disease outcome. Furthermore my research examined endogenous GPR54 expression in breast and ovarian cells to create a knockdown cell line model to further study the autocrine signalling effects of kisspeptin and GPR54 in an epithelial environment.  1.1 Epithelial Kisspeptin Though the kisspeptin encoding gene KiSS1 is now known to be located on chromosome 1 [1], kisspeptin (Kp) was first discovered through microcell- mediated transfer of chromosome 6 [2]. Highly metastatic melanoma tumours have been characterized as missing genomic material from chromosome arm 6q and subsequent replacement of chromosome 6 in a melanoma cell line inhibited  2 metastasis [3, 4]. By comparing gene expression differences between the metastatic parental cell line and the chromosome 6 carrying line, KiSS1 was revealed as a metastasis suppressor gene candidate [2, 5]. Since then, a regulator for KiSS1 expression located on chromosome 6 has yet to be determined although there is evidence CRSP3 (DRIP130), a co-factor for the Sp- 1 transcription factor, may regulate KiSS1 expression through regulation of TXNIP which is also located on chromosome 1 [6]. The KiSS1 gene locus encodes for the kisspeptins, of which four of varying lengths are currently known; kisspeptin-54, kisspeptin-14, kisspeptin-13, and kisspeptin-10, the largest of which (Kp-54) is also known as metastin due to its anti-invasive properties [2, 7, 8]. Prior to processing, the 145 amino acid KiSS1 pro-peptide contains a secretory sequence, two dibasic cleavage sites, and an amidation site [7]. The putative first cleavage site is of unknown significance as a kisspeptin-64 has yet to be observed, however the second dibasic cleavage creates the N-terminus of kisspeptin-54 (Figure 1.1 A, B). These processing sites are conserved in rodents, although the 52aa kisspeptin in mice has only 46% similarity to human Kp-54 [9] (Figure 1.1 C). Both the secretory sequence and the amidation site are necessary for a functional kisspeptin as when either are mutated or omitted Kp is inactive [8, 10]. The enzymes required for this kisspeptin processing have yet to be elucidated, however the matrix metalloproteinases 2 and 9 (MMP2, MMP9) have been shown to cleave and inactivate metastin and Kp-10 at Gly118-Leu119 [11] (Figure 1.1 B).  3      Figure 1.1 The kisspeptins A) The full length pro-peptide for kisspeptin is 145 amino acids (aa) in length, has a 19 aa secretory sequence (black) and three cleavage sites (K57-P), (R67-G), and (R124-E). Full length human Kp-54 is shaded in gray, and the N-terminal becomes amidated losing three amino acids during processing (B). B) The four known human kisspeptins can be cleaved by MMPs at G118-L (red line) and become deactivated. C) The mouse Kp-52 sequence is shown for comparison (Modified from review articles [4, 19]).   4 Although it was determined that Kp can be inactivated by MMP2 and MMP9 [11], both activity [12] and expression [13] of MMP2 was decreased after Kp stimulation. Furthermore, the KiSS1 overexpressing fibrosarcoma HT-1080 cells demonstrated a decrease in MMP9 activity and expression. Inverse correlation between Kp and MMP expression may be relevant to the anti-invasive ability of Kp as MMP activity has been credited with creating a metastatically favourable environment.  There has been some debate among research groups as to whether there are differences in affinity for GPR54 among the Kp peptides. Using competitive binding assays and calcium release asays, it has been claimed that there is no difference among kisspeptins [7], while others have claimed that Kp-10 has the highest affinity for GPR54 [8, 9, 14], and one group has claimed Kp-10 to be the only Kp to elicit a response from endogenous GPR54 in trophoblast cells [12]. However, endogenous Kp-10 was purified from the placenta by only two of these research groups [12, 14] whereas endogenous Kp-54 was always detected. Finally, it has been claimed that Kp-54 has the highest potency in vivo [15, 16] and it is not cleaved into smaller products in human plasma [17, 18]. Since the 145 amino acid pro-peptide kisspeptin sequence has no cleavage sites specific for the smaller peptides, this argues that the smaller peptides may be degraded products of Kp-54. Throughout this dissertation the kisspeptins will be referred to collectively as kisspeptin or Kp unless specification of which kisspeptin is merited. Similarly,  5 the cognate receptor for Kp, GPR54, has had a variety of aliases (AXOR12; HOT7T175; KiSS1R) but will be referred to as GPR54 throughout the text.  1.1.1 Establishing kisspeptin as anti-metastatic Originally discovered to be lost in a highly metastatic melanoma cell line, kisspeptin was initially termed metastin after in vitro and in vivo studies demonstrated that overexpression of KiSS1 led to a less invasive phenotype [2]. Since this key discovery paper subsequent in vitro and in vivo studies conducted in a variety of cell lines have had similar conclusions. The first of these cell lines was a highly metastatic melanoma cell line, C8161, which demonstrated decreased invasiveness upon KiSS1 overexpression as measured by reduced metastases when injected into athymic nude mice as compared to parental controls [2]. This same group went on to transfect a highly invasive breast cancer cell line, MDA-MB-435, and had a similar outcome with reduced metastases and decreased tumour burden [20]. These original KiSS1 overexpressing studies report specifically as Kp being anti-metastatic and not anti-tumourigenic [2, 20]. GPR54, a G-protein coupled receptor of the calcium release Gq family of G-proteins [7, 8, 14] (Figure 1.2) located on chromosome 19p13.3 [21], was discovered as the cognate receptor for Kp simultaneously by three groups [7, 8, 14]. Ohtaki et al [8] had similar findings as Lee et al. [2, 20] by stimulating GPR54 overexpressing cells with Kp. Using CHO and mouse melanoma B16-BL6 cells, there was a decrease in in vitro invasion and migration as well as decreased in vivo metastases, however proliferation was not specifically assessed at this time.   6     Figure 1.2 GPR54 intracellular signalling through the Gq pathway Upon Kp binding to GPR54 it is believed that intracellular signalling occurs through a Gq pathway. After stimulation, the G-protein G disassociates from the G-proteins  and . G gains another phosphorylation group through guanosine triphosphate (GTP) conversion to guanosine diphosphate (GDP). The triphospate G protein activates phospholipase C (PLC) which cleaves phosphatidylinositol 4,5-bisphosphate (PIP2) into inositol 1,4,5-triphosphate (IP3) and diacyl glycerol (DAG). DAG and calcium released from internal stores through IP3 binding of calcium channels on the endoplasmic reticulum go on to  7 activate protein kinase C (PKC). Among other targets, PKC has the ability to phosphorylate Raf-1 thus stimulating the mitogen-activated protein kinase (MAPK) pathway leading to phosphorylation of MAPK kinase (MEK) followed by phosphorylation of extracellular signal-regulated kinases (pERK).  8  In a follow-up study by this same group, a proliferation assay was conducted using the same GPR54 transfected CHO cell line and growth was inhibited with the addition of Kp to the cell culture media [22]. Moreover, Kotani et al. [7] assessed proliferation of GPR54 overexpressing CHO cells stimulated with Kp and also determined a decrease in proliferation, though the MAP kinase pathway was activated. They did not determine the invasive or metastatic ability of these cells. Stress fiber activation was witnessed by both groups and considered as a possible anti-metastatic mechanism of action. Since these original discovery papers, there have been conflicting reports of whether stimulation of GPR54 inhibits proliferation as part of the anti- mestastatic behaviour of Kp. Initial studies made the distinction of Kp overexpression specifically having no effect on proliferation while reducing metastatic burden [2, 20]. However, after the discovery of GPR54 as the canonical receptor for Kp, overexpression of GPR54 was used as an alternative to KiSS1 overexpression to study anti-metastatic behaviour. Although proliferation was assessed in both cell line models, only GPR54 overexpressing cells inhibited proliferation. In addition to the studies that have already been examined, further reports are discussed below.  Overexpression of KiSS1 in the SKOV3 ovarian cell line expressing endogenous GPR54 led to decreased in vitro migration and colony formation as well as decreased in vivo metastasis formation, but did not affect cell proliferation [23]. In a fibrosarcoma cell line, HT-1080, overexpression of KiSS1 also led to decreased migration without having an effect on proliferation [24]. Another group  9 created a stably overexpressing GPR54 MDA-MB-435 cell line after discovering that endogenous GPR54 was missing from these breast cancer cells. However, unlike the KiSS1 overexpressing studies, they report a decrease in cell proliferation and colony formation upon Kp stimulation of these transfected cells [25]. Furthermore, GPR54 was overexpressed in a human papillary thyroid carcinoma cell line and upon Kp stimulation there was inhibition of migration and proliferation [26]. Alternative to creating overexpressing cell lines is to exploit those that have high endogenous levels of either GPR54 or Kp and compare them to cell lines with lower levels. Two GPR54 expressing renal cell carcinoma cell lines of varying Kp expression were compared and determined to have opposite migratory phenotypes upon Kp stimulation. The KU19-20 cells without any detectable Kp mRNA, had decreased migratory behaviour upon Kp stimulation while the highly Kp expressing Caki-1 cells did not. Neither cell line showed a decreased in proliferation upon external Kp stimulation [13, 27]. Similarly, no effect on cell proliferation was seen when Kp was added to the cell culture media of the pancreatic cell lines PANC-1 and AsPc-1 although there was an increase in pERK. However, the highly GPR54 expressing PANC-1 demonstrated a decrease in migration while no effect was seen with the AsPc-1 that expresses GPR54 at lower levels and high levels of Kp as compared to PANC-1. Nor was there a significant difference in the invasive ability of these two lines [28]. Both ligand and receptor are highly expressed in the placenta, which can be used as a model for controlled invasion [8, 14]. To assess epithelial GPR54  10 and Kp in normal physiology, first term trophoblast explant tissue was used to study the effects of Kp on cells with endogenous GPR54. Upon Kp-10 stimulation of this tissue, extravillous outgrowth ceased, and this was determined to be an effect of migration arrest and not due to decreased proliferation [12]. Currently it is unknown why GPR54 overexpressing cell lines uniquely induce an anti-proliferative phenotype as compared to other cell line models that have been used to study Kp-GPR54 signalling. Regardless, all models have demonstrated that GPR54 activation through Kp stimulates anti-invasive and anti-metastatic behaviour.  1.1.2 Kisspeptin in clinical samples As Kp and GPR54 have anti-metastatic ability, loss of either ligand or receptor in human tumour samples would be expected to correlate with a more invasive phenotype. Similarly, the gain of either ligand or receptor would be expected to correlate with a less invasive phenotype. The difficulty that has been encountered in supporting these hypotheses has been the manufacture of reliable antibodies, as perhaps is the difficulty with any novel peptide. As an example, it has been recently reported that a human Kp antibody, previously believed to be specific, cross-reacts with the human neuropeptide FF [29]. This RF-amide peptide is primarily expressed in the central nervous system, however it can also be measured in human plasma [30, 31]. To overcome this antibody issue, mRNA in situ hybridization and quantitative real-time PCR have been employed in a number of studies. Expression of the ligand and receptor in  11 tumours has been compared to a variety of patient characteristics to establish whether KiSS1 and/or GPR54 expression correlate with invasion. Specifically, gene expression has been correlated with tissue invasive phenotype (normal and metastatic), clinicopathological markers (grade and stage), and patient survival (disease recurrence and overall survival).  With the discovery of GPR54 as the receptor for Kp, the expression of both ligand and receptor were initially assessed in a small panel of tumours of various tissue type: four breast, two uterus, five ovary, one small intestine, and two colon. These tumours generally had increased levels of GPR54 as compared to their normal adjacent tissue while KiSS1 expression seemed to be lacking in both tumours and in normal tissue [8]. GPR54 is overexpressed in papillary thyroid cancers as compared to follicular carcinomas or normal tissues, but due to small sample size no significant correlations could be determined [32]. In renal cell carcinoma it was demonstrated by real-time PCR and IHC that higher levels of GPR54 were expressed in cancer compared to normal tissue while KiSS1 was similarly expressed in both normal renal tissue and disease [27]. Conversely, in the normal pancreas the average KiSS1 expression is higher than the average expression in tumour samples while the opposite is true for GPR54 [28].  Several research groups used clinical samples with linked outcome data allowing for comparison of KiSS1 and GPR54 expression with prognosis. In gastric cancer, downregulation of KiSS1 is significantly associated with venous invasion and disease recurrence. Furthermore, loss of expression correlates with poor prognosis in overall and disease specific survival independent of stage [33].  12 Similarly, in esophageal squamous cell carcinoma loss of either one or both GPR54 and KiSS1 mRNA correlates significantly with poor prognosis in disease specific survival and correlates independently with lymph node metastasis [34]. For bladder cancer, loss of KiSS1 expression significantly correlates with poor prognosis in overall survival [35]. There are two reports of KiSS1/GPR54 expression in clinical cases that equate expression as a poor prognostic marker. In hepatocellular cancer, overexpression of KiSS1 and GPR54 as determined by quantitative PCR correlated significantly with advanced stage that is a marker for poor prognosis [36]. These results have since been confirmed using IHC where Kp was an independent prognostic marker for poor prognosis in both disease specific and overall survival [37]. As this study used the Kp polyclonal rabbit antibody that has since been shown to be non-specific, the validity of these results are questionable. Additionally, in a panel of 124 breast cancers, KiSS1 expression correlated with poor prognosis in disease specific survival [38]. However, the real-time PCR was normalized to cytokeratin-19 (CK-19) expression within the same tumour sample. CK-19 has variable expression throughout breast tumours and is itself a marker for poor prognosis [39]. For those studies without outcome data, expression of KiSS1 and/or GPR54 in invasive disease was compared to more benign phenotypes. In melanoma, early skin cancer lesions and benign cases all expressed KiSS1 as compared to the larger lesions and metastatic disease that exhibited KiSS1 expression in only half of the cases [40]. Furthermore, cases with loss of KiSS1  13 expression significantly correlated with loss of heterozygosity (LOH) on chromosome 6 (q16.3-23), the region believed responsible for Kp regulation [40]. In brain metastases arising from breast cancer, KiSS1 expression was lost compared to the primary lesion [41]. These two studies have similar results as those with linked clinical outcome; they demonstrate that loss of KiSS1 correlates with a more invasive disease. When assessing KiSS1 and GPR54 expression in clinical samples, the distinction between anti-tumourigenic and anti-metastatic is important to consider. Many tumours, across a variety of tissues, express increased KiSS1 and GPR54 expression as compared to their normal counterparts, which may intuitively suggest that Kp and GPR54 are not representative of favourable prognosis. However, when KiSS1 and GPR54 expression are linked with clinical outcome data, expression does correlate with favourable prognosis in almost all clinical samples that have been studied to date. This observation is supported by those studies that compare loss of KiSS1 expression in metastatic lesions with primary tumours. Prognosis is an important indicator of invasive disease, as most cancers are not necessarily lethal as primary tumours; it is often the metastatic ability and invasiveness of cancer that leads to death of the patient [42, 43]. As KiSS1 and GPR54 are expressed more highly in tumours compared to normal tissues, yet lost in more invasive disease, this supports the hypothesis that Kp- GPR54 are anti-metastatic and not anti-tumourigenic. Table 1.1 summarizes all the statistically significant results exploring KiSS1 and GPR54 expression in clinical cases as discussed above.  1 4  Table 1.1 Summary of statistically significant KiSS1 and GPR54 expression in clinical cases Exp=expression, DSS=disease specific survival, OS=overall survival  Malignancy Gene(s) Analyzed Cases Assay Used Comparison Significant Correlation Survival Analysis Multivariable Analysis Reference Gastric Adenocarcinoma KiSS1 40 RNase protection Compared to adjacent normal mucosa Loss correlates with venous invasion Loss correlates with DSS and OS Independent marker Dhar, 2004 Ductal and Lobular Breast Cancer KiSS1 and GPR54 124 QPCR QPCR normalized to CK-19 KiSS1 exp correlates with node positivity KiSS1 exp correlates with DSS Not done Martin, 2005 Hepatocellular Carcinoma KiSS1 and GPR54 60 QPCR Compared to 8 normal and 60 cirrohtic livers Exp correlates with higher stage Exp correlates with DSS Not done Ikeguchi, 2003 Brain Metastasis, Primary Breast KiSS1 5 QPCR Compared to 10 primary tumours Loss of KiSS1 in metastases Not done Not done Stark, 2005 Esophageal Squamous Cell Carcinoma KiSS1 and GPR54 71 QPCR Compared to adjacent normal tissue Both inversely correlate with node positivity Loss correlates with DSS Independent marker Ikeguchi, 2004 15 cDNA mircroarray Compared to 9 cell lines Exp correlates with non-invasive tumours Exp correlates with OS Not done Sanchez- Carbayo, 2003 Bladder Transitional Cell Carcinoma KiSS1 25 paired, 173 RNA in situ 25 compared to normal urothelium Loss correlates with venous invasion Exp correlates with OS Not done Sanchez- Carbayo, 2003 Pancreatic Cancer KiSS1 and GPR54 30 QPCR Compared to 5 normal Loss of KiSS1, Exp GPR54 in cancer Not done Not done Masui, 2004 Papillary and Follicular Thyroid Cancer KiSS1 and GPR54 23 QPCR Compared to 11 normal 2 benign Papillary exp increased over follicular Not done Not done Ringel, 2002 Renal Cell Carcinoma KiSS1 and GPR54 25 QPCR Compared to normal renal cortex GPR54 higher in RCC compared to renal cortex Not done Not done Shoji, 2008  15 1.2 Neuroendocrine Kisspeptin It has been well established that sexual reproduction in mammals relies on the activation of the hypothalamic-pituitary-gonadal axis. This axis is mediated by gonadotropin releasing hormone (GnRH) that is released from neurons in the hypothalamus to stimulate gonadotropin release from the anterior pituitary. The gonadotropes release the gonadotropins, lutenizing hormone (LH) and follicular stimulating hormone (FSH), into circulation to act upon the gonads in an endocrine fashion to induce sex hormone release and stimulate gametogenesis [44]. The estrogen and androgen sex hormones then have a negative feedback effect on the hypothalamus that reduces the levels of gonadotropins within the system. For many years the mechanism of this feedback effect was a mystery as the GnRH expressing neurons do not express any of the steroid hormone receptors [45, 46]. Due to this curious circumstance, it was evident that an important regulator in the mammalian reproductive axis was missing.  1.2.1 Kisspeptin regulation of GnRH through GPR54 Kisspeptin was first linked to the hypothalamic-pituitary-gonadal axis by the creation of a Gpr54 knockout mouse and the discovery of an inactivating GPR54 mutation in humans. Both mutated species demonstrated hypogonadotropic hypogonadism; the absence of sexual maturation due to decreased gonadotropins in circulation [47]. Since this groundbreaking discovery, GnRH  16 regulation through Kp release has been studied in many species including the lower vertebrates [48-50]. Activation of the hypothalamic-pituitary-gonadal axis has been determined by GnRH release with accompanying LH and FSH increase upon Kp stimulation in; mice [51, 52], rats [53-59], sheep [52, 60], monkeys [61, 62] and humans [17, 18]. This kisspeptin activity has been demonstrated to be effective both peripherally and centrally and is GPR54 specific [52, 63, 64]. GPR54 has been colocalized to GnRH neurons in the hypothalamus [52, 54], and subsequent activation of GPR54 by kisspeptin on these neurons results in GnRH neuronal activity [55, 59, 65, 66] that is concentration dependent [67] and directly releases GnRH [52, 68, 69] that can be inhibited by a GnRH antagonist [51, 54, 55, 57, 61, 62]. Though GPR54 is highly expressed in the pituitary, Kp does not seem to promote the release of the gonadotropins from the pituitary directly. In rats, pituitary explants and primary cell cultures stimulated with Kp did not have an effect on LH or FSH release [55, 59]. One group did show LH release from pituitary tissues at a much lower level than GnRH and without a similar effect on FSH [56, 57]. Conversely, LH was released from primary rat pituitary cells at higher concentrations upon Kp stimulation than when GnRH was used, but this was sex specific to males. Furthermore, calcium flux was also witnessed in a small subset of the pituitary cells (~10%) in this study, the authors believing these cells to be the somatotrophs and gonadotrophs [70].  17 In the ewe, though there was release of LH from the primary culture of pituitary cells cultured from the follicular phase after Kp stimulation in vitro, this behaviour was absent in in vivo studies [71]. The trend seems to be that there is only an effect when the pituitary is reduced to a single cell layer, but not while it is in its native three-dimensional state.  1.2.2 Hormonal feedback regulation mediated by Kp and GPR54 in rodents In murine models, Kiss1 is expressed in the anteroventral periventricular nucleus (AVPV) and the arcuate nucleus (ARC) in the hypothalamus. This has been determined by immunohistochemical (IHC) analysis and RNA in situ hybridization [51, 72-74]. These neuron processes lie adjacent to GnRH neuron bodies suggesting their direct effect on GnRH release while not being co-localized directly with the GnRH neuron [75], as is the case for the GPR54 receptor [52, 55]. Furthermore, estrogen receptor alpha (ER) has been localized directly in the Kiss1 expressing neurons [73, 74] without being co-localized to the GnRH neurons [76]. Prior to the discovery of Kp and GPR54, the lack of ER on GnRH neurons while still being affected by estrogen positive and negative feedback had been a mystery. Due to the position of Kiss1 expressing neurons and ER, it is thought that the Kp neurons mediate the feedback effects on the hypothalamus. Within the male mouse, Kiss1 expression is upregulated in the ARC after castration [74]. This effect can be reversed by implantion of testosterone that seems to be relayed through the Kiss1 expressing neurons through both ER  18 and the androgen receptor (AR). Dihydrotestosterone (DHT) only reduced Kiss1 expression by half in castrated mice while both testosterone and estrogen replacement completely reduced the elevated Kiss1 expression [74]. Unlike DHT, testosterone can be aromatized to estrogen suggesting that both the AR and ER were in use. This change in Kiss1 expression due to castration was still observed in the ERKO and non-functional AR mice suggesting that testosterone regulates Kiss1 expression either through the AR or through aromatization of testosterone and the ER [74]. Similarly, ovarectomized female mice have an increase in Kiss1 expression within the ARC that is reduced by estrogen implants [77]. However, the AVPV in female mice seems to regulate the positive feedback effect necessary for the LH surge, as there is a decrease in Kiss1 expression within the AVPV in ovariectomized mice that is increased by estrogen replacement [73]. The ER expressing Kiss1 neurons in the AVPV also express the progesterone receptor [72] that is necessary for the LH surge required for female ovulation and GnRH activity at the time of the LH surge [72].  In male and female rats, Gpr54 and Kiss1 RNA expression in the hypothalamus was maximal at puberty compared to postnatal, prepubertal and adult rats [78]. In castrated males, Kiss1 expression was elevated and this effect was abrogated with testosterone replacement while in females a similar effect was noticed with estrogen replacement in ovariectomized rats [78, 79]. Continous Kp stimulation in rats showed desensitization of the axis by a decrease in LH  19 overtime although GnRH RNA levels did not decrease overtime, suggesting that Kp and GPR54 have a role in GnRH release, not expression [57] Specifically, Kiss1 is highly expressed in the ARC as determined by QPCR and IHC and these neurons also express ER [79]. Female ovariectomized rats have decreased Kp expression in the AVPV as compared to intact females. Further, estrogen replacement in ovariectomized rats increased Kiss1 expression in the AVPV [80, 81]. Comparatively, intact male rats have very few Kiss1 neurons and subsequently very little Kiss1 expression in the AVPV [80, 81]. However, Kp neurons were highly expressed in the ARC of both gonadectomized male and female rats, and these levels were reduced with the presence of hormonal implants [80, 81]. In female rats, Kiss1 and GPR54 had maximum expression during the diestrus phase of the estrus cycle [78, 79]. This change in Kiss1 expression throughout the estrus cycle was variable depending on which region of the hypothalamus was being studied. In the AVPV, Kiss1 had the highest expression during proestrus and the lowest expression at diestrus, yet in the ARC Kiss1 had the highest expression at diestrus and lowest in proestrus [80]. Intact female rats failed to undergo ovulation or have a LH increase when pretreated with ER antagonist even after intracerebral injection with Kp-10. This was compared to LH release and ovulation after GnRH treatment [82]. No such effect was reported when mice were pretreated with an ER antagonist prior to injection, and only a modest decline in LH was seen after progesterone receptor  20 (PR) antogonist pretreatment. Again, this effect was not witnessed in the GnRH injected rats [82]. Similarly, PR and ER were required in a normal cycling female treated with empty vehicle for FSH release for the preovulatory phase as was seen in the LH release studies [83]. While both ER and PR antagonists blocked FSH release stimulated by Kp-10, as seen for LH, there was also a decrease in FSH release with ER. For GnRH injections which were unaffected by ER, ER, PR antagonist pretreatment in the release of LH, ER and PR antogonists decreased FSH release after GnRH stimulation [83].  To summarize the above scientific findings; the AVPV is recognized as the hypothalamic region responsible for positive feedback and the subsequent LH surge required for ovulation, while the ARC is responsible for the negative feedback within the Kp-GPR54 regulated hypothalamic-pituitary-gonadal axis in rodents (Figure 1.3).  1.2.3 Hormonal regulation mediated by Kp and GPR54 in primates In monkeys, when human Kp-10 was continuously administered over a 4 day period there was an initial spike in LH secretion, followed by LH returning to normal levels after the first day of continuous infusion. By the fourth day, although N-methyl-D-aspartate acid (NMDA) and GnRH could elicit a LH surge, there was no response from a bolus Kp-10 injection. However, 21 hours after cessation of continuous Kp stimulation, LH was once again responsive to a single bolus of Kp-10 [84]. Further, Kp is released in a pulsatile fashion similar to  21 that of GnRH (LHRH-1) [69]. Kp is also increased in the stalk-median eminance of midpubertal monkeys as compared to prepubertal and early pubertal monkeys [69]. KiSS1 has been localized to the ARC of the medial basal hypothalamus (MBH) of the castrated male monkey [85, 86]. Assessing hormonal feedback by testosterone implant into castrated males found that KiSS1 expression was decreased in the MBH with testosterone treatment while KiSS1 RNA was low in the preoptic area (POA) regardless of treatment. GPR54 RNA levels were unaffected for this study [86]. Comparatively, KiSS1 expression was increased in the infundibular nucleus of the MBH of ovariectomized female monkeys [87, 88] as compared to the estrogen replacement group of animals [88]. However, there was no change in expression found in the preoptic area for either species [87, 88]. The primate infundibular nucleus has been equated to the arcuate nucleus (ARC) in other species, the site for Kp negative feedback [89, 90] and the preoptic area equated to the anteroventral periventricular (AVPV) found in rodents [88]. Menopause in human females can be used as a castration comparison model as has been done in experimental animals [91]. Using neuronal tissue obtained from deceased women, there was an increase in KiSS1 mRNA expression in the infundibular nucleus of post-menopausal woman as compared to pre-menopausal and this was reflected in postmenopausal female monkeys [88]. This suggests that there is a similar negative feedback loop affecting KiSS1 expression in humans; however further studies are required.  22   Figure 1.3 Kp and GPR54 hormonal feedback regulation in the rodent Within the hypothalamus the arcuate nucleus (ARC) mediates the negative feedback through Kp expressing neurons in both male and female rodents. The anteroventral periventricular nucleus (AVPV) is specific to female positive feedback regulation required for ovulation. Kp is released to stimulate GPR54 located on the GnRH neuron also within the hypothalamus that then signals to the pituitary to release the gonadotropins, LH and FSH that then go on to stimulate sex hormone release from the gonads (Modified from reviews [92-94]).   23 1.2.4 GPR54/Kisspeptin mutations in humans The discovery of a homozygous single nucleotide mutation in exon 3 (L148S) of GPR54 in 6 individuals with idiopathic hypogonadotropic hypogonadism (IHH) from a consanguineous family, led to the discovery of kisspeptin’s significant role in stimulating the endocrine cascade [47]. In the same publication, an IHH patient was discovered to be a compound heterozygote, with one allele coding for a truncating mutation (R331X) and the other allele with a nonstop mutation (X399R), both in exon 5. Functional studies revealed that there was a decrease in GPR54 activity in the L148S point mutation by 65% and a decreased by 67% for the R331X premature stop codon. Since the nonstop mutation X399R affected GPR54 conformation, it could not be used for functional studies, but the simple addition of arginine to the end of GPR54 was enough to reduce activity by 61%. Simultaneously, a homozygous deletion also in exon 5 was found to cause IHH in a consanguineous family with five affected children [95]. This deletion leads to a reading frame shift resulting in an altered protein sequence beginning from the third intracellular loop of GPR54. It is as yet unknown if this deletion leads to complete loss of expression of this protein or simply dysregulation of GPR54 function. Since these initial discoveries, other mutations have since been reported. Two of these mutations were found in one isolated hypogonadotropic hypogonadism patient; a transversion in exon 4 from the T nucleic acid to C resulting in a C223R change in amino acid sequence and a transversion in exon 5 G>T resulting in an R297L sequence change thus creating a compound  24 heterozygote. The C223R mutation was found to have a more significant decrease in GPR54 activity (80%) than the R297L (15%). Both the asymptomatic mother and brother of the proband were heterozygous for the R297L mutation [96]. Another frameshift mutation, arising from a single nucleotide insertion in exon 5, created the IHH phenotype in a homozygous patient [97]. This insertion results in an elongated 441 amino acid protein sequence for GPR54 as compared to the normal 398 amino acids. There were no functional studies to assess potential decrease of GPR54 activity in this case, although due to a probable conformation change that would arise from the elongated protein it is most likely that these studies are not possible. The most recent report of a GPR54 mutation resulting in IHH was published last year. A single homozygous point mutation in exon 2 (L102P) was found in 5 patients from two separate consanguineous families resulting in decreased Kp binding affinity and complete loss of GPR54 signal [98]. GPR54 signaling through Kp binding activates calcium flux specific for the G-protein coupled receptors of the Gq subfamily [7, 8, 14]. GPR54 activity can thus be measured by calcium release assays, inositol phosphatase (IP3) activity and has been elucidated to signal through the MAP kinase pathway allowing upregulation of pERK to also be used as a marker for GPR54 activity [7, 24, 28, 32]. By assessing at which point GPR54 activity is lost, one could hypothesize or elucidate how the mutation is affecting downstream events. For the L102P point mutation affecting the first extracellular loop of GPR54, decreased Kp binding  25 was determined to be the consequence of the mutation. For the deletion and insertion resulting in a frameshift and elongated GPR54, it would be reasonable to assume that an elongated protein would result in a conformation change that would target the receptor for the ubiquitin-proteasome-degradation pathway and result in the loss of the receptor. Similarly, the R331X point mutation giving rise to a stop codon may result in a truncated protein being targeted for degradation, or perhaps the intracellular portion required for signaling is lost. The second extracellular mutation R297L may also result in loss of Kp affinity for GPR54, however it seems this is a much milder affect than the L102P mutation since there is only 15% decrease in GPR54 activity. The second mutation in this compound heterozygote, C223R, [99] results in a change of charge from the polar cysteine to the positive arginine. This strong positive charge may be too closely positioned to the hydrophobic plasma membrane resulting in a displaced intracellular loop. The first in-depth functional analysis of one of the GPR54 loss-of-function mutations was recently published [100]. By overexpressing the L148S mutation with an epitope tag and through the completion of a series of functional studies, it was determined that it is the second intracellular loop of GPR54 that is specific for G protein coupling to the receptor. The loss of GPR54 signalling was due to the inability of GPR54 to undergo a conformational change allowing the G protein to dissociate from the G subunit, thus arresting the signalling cascade and not due to the inability of G protein to interact with GPR54.  Further, they  26 determined that the overexpression of the Gq subunit partially rescued the phenotype. All the aforementioned mutations are loss of function mutations. Recently, the first activating GPR54 mutation was published [99]. An 8 year old female presenting with precocious puberty was found to have a single heterozygous point mutation from C>G resulting into an amino acid change from arginine to proline. This mutation led to prolonged GPR54 signalling with a longer response by the MAP kinase pathway. It is interesting to note that the activating mutation is intracellular rather than extracellular. Extracellular over-activation of GPR54 such as increased Kp binding has been shown to desensitize GPR54 [68, 84]. There has yet to be discovered an inactivating or activating KiSS1 mutation. However, the creation of Kiss1 knockout mice recapitulated the phenotype expressed by the Gpr54 knockouts. All knockout mice for either Gpr54 or Kiss1 have resulted in the same loss-of-sexual-maturation phenotype [47, 63, 64, 101, 102].  Figure 1.4 summarizes all the human genomic mutations resulting in either loss of GPR54 function or gain of function that have been discussed.  1.2.5 Kiss1 and Gpr54 knockout mouse models There are currently four Gpr54 knockout mouse models [47, 63, 64, 102], and two Kiss1 knockout mice that have been created [64, 101]. All six of the knockout mice have the same hypogonadotrophic hypogonadism phenotype as seen in GPR54 mutated humans. The males have small testes, microphallus and smaller  27 body weights, while the females have smaller ovaries, threadlike uteri, and delayed vaginal openings indicative of a lack of sexual maturation. Furthermore, these mice are sterile due to disruption of oogenesis and spermatogenesis. The Gpr54 knockout mice also fail to demonstrate normal sexual behaviour, but this can be reversed with sufficient sex steroid hormone replacement [103].  The knockout mice exhibit low levels of the gonadotropins, LH and FSH, as well as low levels of sex steroids; testosterone in males and 17--estradiol in females. Gnrh expression within the hypothalamus of the knockout mice is similar to that of wildtype mice indicating that loss of Gnrh expression is not the mechanism behind the low gonadotropin levels, but more likely due to the inability of GnRH to be released [47, 52, 101]. As Gpr54 is intact in the Kiss1 knockout mice, introducing Kp back into the system of these mice releases the gonadotropins [64, 101]. This is not the case for the Gpr54 knockout mice demonstrating that the hypothalamic-pituitary axis is specifically regulated through GPR54.    28     Figure 1.4 Summary of human GPR54 mutations and their effects Green circles represent inactivating mutations while the green star represents the activating mutation. Red amino acids represent the specific deactivating mutation within the protein sequence while the green amino acid represents the activating mutation. Pink text and percentage represents the effect of the mutation and by how much the activity of GPR54 has been affected when known.  represents conformational change, downward arrows represent decrease in either Kp binding or GPR54 production, upward arrow represent prolonged pERK activity (Modified from review article [104]).   29 1.2.6 Circulating kisspeptins Kp was discovered as the ligand for GPR54 after being isolated from placental fractions and both ligand and receptor are highly expressed in the placenta [2, 7, 8, 14]. Originally, Kp and GPR54 were found to be more highly expressed in early pregnancy as compared to term placentas [12, 105], but this has since been refuted [106, 107]. Kp and GPR54 have decreased expression in more invasive choriocarcinoma cell lines as compared to healthy placenta or molar pregnancies [105]. Since Kp has the protein sequence of a secreted peptide, it follows that one would try to measure Kp in a blood sample. Although Kp is barely measurable in men and non-pregnant women, it is at 10,000 fold higher in plasma of pregnant women, reaching its highest levels by the third trimester of pregnancy [106, 107]. It is also elevated in the umbilical cord blood of newborns though not as greatly as the maternal plasma [108]. Although, Horikoshi et al. [107] showed by RNA in situ hybridization and IHC that Kp is expressed in term placenta, circulating Kp quickly falls back to normal levels after birth. The ability to measure Kp in blood samples led to the exploration of Kp as a serum biomarker for disease. Small for gestational age babies (SGA) had lower levels of metastin in their mother’s serum as compared to an equal number of control normal pregnancies [109]. -HCG levels suggested that the SGA was not due to a decrease in placental size and therefore a possible cause of SGA could be a decreased in invasive ability of the placenta. This would support the role of Kp and GPR54 as regulators of trophoblast invasion during pregnancy. With respect to cancer, Kp was measured in plasma of women with gestational trophoblastic neoplasia, a cancer of placental origin, and were shown  30 to have increased Kp [106]. This increased Kp dropped back to normal physiological levels once the tumour had been removed surgically and the patient had undergone chemotherapy. The disadvantage in this instance is that hCG is already an establish serum marker for the diagnosis and follow-up of this disease. Since many tumours have been documented to express Kp, it is surprising there have not been more publications demonstrating increased Kp levels in cancer patients.  One would assume that since it is a normal physiological event to have extremely high levels of Kp in serum during pregnancy that there should be no adverse consequence to this phenomenon. In 2005 the first study to inject Kp into humans was published. The study showed that upon intravenous injection of Kp into healthy adult male volunteers, LH, FHS and testosterone levels increased while sex hormone binding globulin (SHBG) and Inibin B levels were unaffected. Most importantly there were no reports of any side effects resulting from the treatment [17]. A similar study was conducting in pre-menopausal healthy females with Kp injected as a single bolus in the abdomen as compared to the iv infusion in men.  Plasma kisspeptin levels do not vary throughout the menstrual cycle, however with the injection of Kp there were increased levels of LH and FSH, but the most significant increase for both gonadotropins occurred during the preovulatory phase. Estrogen and progesterone were significantly increased after Kp injection in the luteal phase only [18]. Again there were no adverse affects from the Kp injection.  31  Since the human injection studies, the same group determined that Kp injection into male rats causes testicular degeneration [15]. Originally thought to be a consequence of prolonged exposure to high Kp levels, it was recently determined that a single peripheral bolus of Kp could cause testicular damage as early as 12 hours post injection. Interestingly, no other tissues were harmed and a similar effect is seen when rats are injected with GnRH [110]. This effect was prevented when mice were pretreated with a GnRH receptor antagonist, suggesting these adverse effects were a result of overstimulation of the hypothalamic-pituitary-axis. This raises the importance of properly assessing suitable Kp concentrations when considering any further human injections, as testicular degeneration could be avoided with still maximal gonadotropin release by adjusting the dose of Kp.  1.3 Hypotheses and Specific Aims 1.3.1 Hypotheses (i) Expression of GPR54/kisspeptin in epithelial malignancies is predictive of disease outcome and (ii) altering endogenous GPR54 signalling in malignant epithelial cells could alter their metastatic properties.  1.3.2 Specific Aims (i) The first aim was to determine the clinical significance of Kp and GPR54 expression in cancer. Although there is extensive literature documenting GPR54  32 and KiSS1 RNA expression in a variety of tumours, there have been few publications based on the immunohistochemical analysis of these two proteins. These results are presented in Chapters 2 and 3. Chapter 2 focuses on the exploration of Kp and GPR54 expression across a panel of cancers while Chapter 3 focuses specifically on Kp and GPR54 expression in ovarian cancer and the related clinical outcome of these patients.  Following the discovery that Kp and GPR54 could be detected in a variety of tumours, we set out to determine if Kp overexpression could then be measured in the plasma of cancer patients. Chapter 4 discusses these results and further explores the initial findings from Chapter 3.  (ii) The second aim was to find breast and/or ovarian cell lines endogenously expressing and signalling through GPR54, to examine the consequences of abrogating epithelial signalling. Previous studies on the anti-metastatic effects of Kp-GPR54 signalling in malignant cells have not addressed endogenous signalling in breast or ovarian cancer. Chapter 5 addresses these findings, with respect to GPR54 expression and signalling in breast and ovarian cells lines, and the issues we encountered while trying to establish this model.  (iii) The third and final aim was to uncover gene loci whose regulation may depend directly on GPR54 signalling in a physiological context. The original objective was to determine whether there are common genes regulated by GPR54 signalling in malignant cells, in comparison with physiological GPR54  33 signalling in the hypothalamus. To discover the hypothalamic gene loci, we conducted differential transcript analysis of the GPR54 and kisspeptin knockout mice hypothalamus. Chapter 6 describes candidate GPR54 regulated genes that were discovered from an Affymetrix and large-scale real-time PCR analysis of microdissected hypothalamic tissue. This chapter also addresses experimentally which transcripts are hormonally regulated and which are independent of hormonal action.    34 1.4 References 1. West A, Vojta PJ, Welch DR, Weissman BE: Chromosome localization and genomic structure of the KiSS-1 metastasis suppressor gene (KISS1). In: Genomics. vol. 54; 1998: 145-148. 2. Lee JH, Miele ME, Hicks DJ, Phillips KK, Trent JM, Weissman BE, Welch DR: KiSS-1, a novel human malignant melanoma metastasis- suppressor gene. J Natl Cancer Inst 1996, 88(23):1731-1737. 3. Harms JF, Welch DR, Miele ME: KISS1 metastasis suppression and emergent pathways. Clin Exp Metastasis 2003, 20(1):11-18. 4. Nash KT, Welch DR: The KISS1 metastasis suppressor: mechanistic insights and clinical utility. Front Biosci 2006, 11:647-659. 5. Lee JH, Welch DR: Identification of highly expressed genes in metastasis-suppressed chromosome 6/human malignant melanoma hybrid cells using subtractive hybridization and differential display. In: Int J Cancer. vol. 71; 1997: 1035-1044. 6. Goldberg SF, Miele ME, Hatta N, Takata M, Paquette-Straub C, Freedman LP, Welch DR: Melanoma metastasis suppression by chromosome 6: evidence for a pathway regulated by CRSP3 and TXNIP. In: Cancer Res. vol. 63; 2003: 432-440. 7. Kotani M, Detheux M, Vandenbogaerde A, Communi D, Vanderwinden JM, Le Poul E, Brézillon S, Tyldesley R, Suarez-Huerta N, Vandeput F et al: The metastasis suppressor gene KiSS-1 encodes kisspeptins, the natural ligands of the orphan G protein-coupled receptor GPR54. In: J Biol Chem. vol. 276; 2001: 34631-34636. 8. Ohtaki T, Shintani Y, Honda S, Matsumoto H, Hori A, Kanehashi K, Terao Y, Kumano S, Takatsu Y, Masuda Y et al: Metastasis suppressor gene KiSS-1 encodes peptide ligand of a G-protein-coupled receptor. Nature 2001, 411(6837):613-617. 9. Terao Y, Kumano S, Takatsu Y, Hattori M, Nishimura A, Ohtaki T, Shintani Y: Expression of KiSS-1, a metastasis suppressor gene, in trophoblast giant cells of the rat placenta. In: Biochim Biophys Acta. vol. 1678; 2004: 102-110. 10. Nash KT, Phadke PA, Navenot JM, Hurst DR, Accavitti-Loper MA, Sztul E, Vaidya KS, Frost AR, Kappes JC, Peiper SC et al: Requirement of KISS1 secretion for multiple organ metastasis suppression and  35 maintenance of tumor dormancy. J Natl Cancer Inst 2007, 99(4):309- 321. 11. Takino T, Koshikawa N, Miyamori H, Tanaka M, Sasaki T, Okada Y, Seiki M, Sato H: Cleavage of metastasis suppressor gene product KiSS-1 protein/metastin by matrix metalloproteinases. Oncogene 2003, 22(30):4617-4626. 12. Bilban M, Ghaffari-Tabrizi N, Hintermann E, Bauer S, Molzer S, Zoratti C, Malli R, Sharabi A, Hiden U, Graier W et al: Kisspeptin-10, a KiSS- 1/metastin-derived decapeptide, is a physiological invasion inhibitor of primary human trophoblasts. J Cell Sci 2004, 117(Pt 8):1319-1328. 13. Yoshioka K, Ohno Y, Horiguchi Y, Ozu C, Namiki K, Tachibana M: Effects of a KiSS-1 peptide, a metastasis suppressor gene, on the invasive ability of renal cell carcinoma cells through a modulation of a matrix metalloproteinase 2 expression. In: Life Sci. 2008. 14. Muir AI, Chamberlain L, Elshourbagy NA, Michalovich D, Moore DJ, Calamari A, Szekeres PG, Sarau HM, Chambers JK, Murdock P et al: AXOR12, a novel human G protein-coupled receptor, activated by the peptide KiSS-1. J Biol Chem 2001, 276(31):28969-28975. 15. Thompson EL, Murphy KG, Patterson M, Bewick GA, Stamp GW, Curtis AE, Cooke JH, Jethwa PH, Todd JF, Ghatei MA et al: Chronic subcutaneous administration of kisspeptin-54 causes testicular degeneration in adult male rats. In: Am J Physiol Endocrinol Metab. vol. 291; 2006: E1074-1082. 16. Tovar S, Vazquez MJ, Navarro VM, Fernández-Fernández R, Castellano JM, Vigo E, Roa J, Casanueva FF, Aguilar E, Pinilla L et al: Effects of single or repeated intravenous administration of kisspeptin upon dynamic LH secretion in conscious male rats. In: Endocrinology. vol. 147; 2006: 2696-2704. 17. Dhillo WS, Chaudhri OB, Patterson M, Thompson EL, Murphy KG, Badman MK, McGowan BM, Amber V, Patel S, Ghatei MA et al: Kisspeptin-54 stimulates the hypothalamic-pituitary gonadal axis in human males. J Clin Endocrinol Metab 2005, 90(12):6609-6615. 18. Dhillo WS, Chaudhri OB, Thompson EL, Murphy KG, Patterson M, Ramachandran R, Nijher GK, Amber V, Kokkinos A, Donaldson M et al: Kisspeptin-54 stimulates gonadotropin release most potently during the preovulatory phase of the menstrual cycle in women. In: J Clin Endocrinol Metab. vol. 92; 2007: 3958-3966. 19. Castano JP, Martinez-Fuentes AJ, Gutierrez-Pascual E, Vaudry H, Tena- Sempere M, Malagon MM: Intracellular signalling pathways activated  36 by kisspeptins through GPR54: do multiple signals underlie function diversity? Peptides 2009, 30(1):10-15. 20. Lee JH, Welch DR: Suppression of metastasis in human breast carcinoma MDA-MB-435 cells after transfection with the metastasis suppressor gene, KiSS-1. Cancer Res 1997, 57(12):2384-2387. 21. Lee DK, Nguyen T, O'Neill GP, Cheng R, Liu Y, Howard AD, Coulombe N, Tan CP, Tang-Nguyen AT, George SR et al: Discovery of a receptor related to the galanin receptors. FEBS Lett 1999, 446(1):103-107. 22. Hori A, Honda S, Asada M, Ohtaki T, Oda K, Watanabe T, Shintani Y, Yamada T, Suenaga M, Kitada C et al: Metastin suppresses the motility and growth of CHO cells transfected with its receptor. Biochem Biophys Res Commun 2001, 286(5):958-963. 23. Jiang Y, Berk M, Singh LS, Tan H, Yin L, Powell CT, Xu Y: KiSS1 suppresses metastasis in human ovarian cancer via inhibition of protein kinase C alpha. Clin Exp Metastasis 2005, 22(5):369-376. 24. Yan C, Wang H, Boyd DD: KiSS-1 represses 92-kDa type IV collagenase expression by down-regulating NF-kappa B binding to the promoter as a consequence of Ikappa Balpha -induced block of p65/p50 nuclear translocation. J Biol Chem 2001, 276(2):1164-1172. 25. Becker JA, Mirjolet JF, Bernard J, Burgeon E, Simons MJ, Vassart G, Parmentier M, Libert F: Activation of GPR54 promotes cell cycle arrest and apoptosis of human tumor cells through a specific transcriptional program not shared by other Gq-coupled receptors. In: Biochem Biophys Res Commun. vol. 326; 2005: 677-686. 26. Stathatos N, Bourdeau I, Espinosa AV, Saji M, Vasko VV, Burman KD, Stratakis CA, Ringel MD: KiSS-1/G protein-coupled receptor 54 metastasis suppressor pathway increases myocyte-enriched calcineurin interacting protein 1 expression and chronically inhibits calcineurin activity. In: J Clin Endocrinol Metab. vol. 90; 2005: 5432- 5440. 27. Shoji S, Tang XY, Umemura S, Itoh J, Takekoshi S, Shima M, Usui Y, Nagata Y, Uchida T, Osamura RY et al: Metastin Inhibits Migration and Invasion of Renal Cell Carcinoma with Overexpression of Metastin Receptor. In: Eur Urol. 2008. 28. Masui T, Doi R, Mori T, Toyoda E, Koizumi M, Kami K, Ito D, Peiper SC, Broach JR, Oishi S et al: Metastin and its variant forms suppress migration of pancreatic cancer cells. Biochem Biophys Res Commun 2004, 315(1):85-92.  37 29. Goodman RL, Lehman MN, Smith JT, Coolen LM, de Oliveira CV, Jafarzadehshirazi MR, Pereira A, Iqbal J, Caraty A, Ciofi P et al: Kisspeptin neurons in the arcuate nucleus of the ewe express both dynorphin A and neurokinin B. In: Endocrinology. vol. 148; 2007: 5752- 5760. 30. Sundblom DM, Kalso E, Tigerstedt I, Wahlbeck K, Panula P, Fyhrquist F: Neuropeptide FF-like immunoreactivity in human cerebrospinal fluid of chronic pain patients and healthy controls. Peptides 1997, 18(7):923-927. 31. Sundblom DM, Panula P, Fyhrquist F: Neuropeptide FF-like immunoreactivity in human plasma. Peptides 1995, 16(2):347-350. 32. Ringel MD, Hardy E, Bernet VJ, Burch HB, Schuppert F, Burman KD, Saji M: Metastin receptor is overexpressed in papillary thyroid cancer and activates MAP kinase in thyroid cancer cells. J Clin Endocrinol Metab 2002, 87(5):2399. 33. Dhar DK, Naora H, Kubota H, Maruyama R, Yoshimura H, Tonomoto Y, Tachibana M, Ono T, Otani H, Nagasue N: Downregulation of KiSS-1 expression is responsible for tumor invasion and worse prognosis in gastric carcinoma. Int J Cancer 2004, 111(6):868-872. 34. Ikeguchi M, Yamaguchi K, Kaibara N: Clinical significance of the loss of KiSS-1 and orphan G-protein-coupled receptor (hOT7T175) gene expression in esophageal squamous cell carcinoma. Clin Cancer Res 2004, 10(4):1379-1383. 35. Sanchez-Carbayo M, Capodieci P, Cordon-Cardo C: Tumor suppressor role of KiSS-1 in bladder cancer: loss of KiSS-1 expression is associated with bladder cancer progression and clinical outcome. Am J Pathol 2003, 162(2):609-617. 36. Ikeguchi M, Hirooka Y, Kaibara N: Quantitative reverse transcriptase polymerase chain reaction analysis for KiSS-1 and orphan G-protein- coupled receptor (hOT7T175) gene expression in hepatocellular carcinoma. J Cancer Res Clin Oncol 2003, 129(9):531-535. 37. Schmid K, Wang X, Haitel A, Sieghart W, Peck-Radosavljevic M, Bodingbauer M, Rasoul-Rockenschaub S, Wrba F: KiSS-1 overexpression as an independent prognostic marker in hepatocellular carcinoma: an immunohistochemical study. In: Virchows Arch. vol. 450; 2007: 143-149. 38. Martin TA, Watkins G, Jiang WG: KiSS-1 expression in human breast cancer. Clin Exp Metastasis 2005, 22(6):503-511.  38 39. Parikh RR, Yang Q, Higgins SA, Haffty BG: Outcomes in young women with breast cancer of triple-negative phenotype: the prognostic significance of CK19 expression. Int J Radiat Oncol Biol Phys 2008, 70(1):35-42. 40. Shirasaki F, Takata M, Hatta N, Takehara K: Loss of expression of the metastasis suppressor gene KiSS1 during melanoma progression and its association with LOH of chromosome 6q16.3-q23. Cancer Res 2001, 61(20):7422-7425. 41. Stark AM, Tongers K, Maass N, Mehdorn HM, Held-Feindt J: Reduced metastasis-suppressor gene mRNA-expression in breast cancer brain metastases. J Cancer Res Clin Oncol 2005, 131(3):191-198. 42. Cannistra SA: Cancer of the ovary. N Engl J Med 2004, 351(24):2519- 2529. 43. Weigelt B, Peterse JL, van 't Veer LJ: Breast cancer metastasis: markers and models. Nat Rev Cancer 2005, 5(8):591-602. 44. Plant TM: Hypothalamic control of the pituitary-gonadal axis in higher primates: key advances over the last two decades. J Neuroendocrinol 2008, 20(6):719-726. 45. Ronnekleiv OK, Kelly MJ: Diversity of ovarian steroid signalling in the hypothalamus. Front Neuroendocrinol 2005, 26(2):65-84. 46. Tilbrook AJ, Clarke IJ: Negative feedback regulation of the secretion and actions of gonadotropin-releasing hormone in males. Biol Reprod 2001, 64(3):735-742. 47. Seminara SB, Messager S, Chatzidaki EE, Thresher RR, Acierno JS, Jr., Shagoury JK, Bo-Abbas Y, Kuohung W, Schwinof KM, Hendrick AG et al: The GPR54 gene as a regulator of puberty. N Engl J Med 2003, 349(17):1614-1627. 48. Lee YR, Tsunekawa K, Moon MJ, Um HN, Hwang JI, Osugi T, Otaki N, Sunakawa Y, Kim K, Vaudry H et al: Molecular evolution of multiple forms of kisspeptins and GPR54 receptors in vertebrates. Endocrinology 2009. 49. Mohamed JS, Benninghoff AD, Holt GJ, Khan IA: Developmental expression of the G protein-coupled receptor 54 and three GnRH mRNAs in the teleost fish cobia. J Mol Endocrinol 2007, 38(1-2):235- 244. 50. Moon JS, Lee YR, Oh da Y, Hwang JI, Lee JY, Kim JI, Vaudry H, Kwon HB, Seong JY: Molecular cloning of the bullfrog kisspeptin receptor  39 GPR54 with high sensitivity to Xenopus kisspeptin. Peptides 2009, 30(1):171-179. 51. Gottsch ML, Cunningham MJ, Smith JT, Popa SM, Acohido BV, Crowley WF, Seminara SB, Clifton DK, Steiner RA: A role for kisspeptins in the regulation of gonadotropin secretion in the mouse. In: Endocrinology. vol. 145; 2004: 4073-4077. 52. Messager S, Chatzidaki EE, Ma D, Hendrick AG, Zahn D, Dixon J, Thresher RR, Malinge I, Lomet D, Carlton MB et al: Kisspeptin directly stimulates gonadotropin-releasing hormone release via G protein- coupled receptor 54. Proc Natl Acad Sci U S A 2005, 102(5):1761-1766. 53. Castellano JM, Navarro VM, Fernandez-Fernandez R, Castano JP, Malagon MM, Aguilar E, Dieguez C, Magni P, Pinilla L, Tena-Sempere M: Ontogeny and mechanisms of action for the stimulatory effect of kisspeptin on gonadotropin-releasing hormone system of the rat. Mol Cell Endocrinol 2006, 257-258:75-83. 54. Irwig MS, Fraley GS, Smith JT, Acohido BV, Popa SM, Cunningham MJ, Gottsch ML, Clifton DK, Steiner RA: Kisspeptin activation of gonadotropin releasing hormone neurons and regulation of KiSS-1 mRNA in the male rat. Neuroendocrinology 2004, 80(4):264-272. 55. Matsui H, Takatsu Y, Kumano S, Matsumoto H, Ohtaki T: Peripheral administration of metastin induces marked gonadotropin release and ovulation in the rat. Biochem Biophys Res Commun 2004, 320(2):383- 388. 56. Navarro VM, Castellano JM, Fernández-Fernández R, Tovar S, Roa J, Mayen A, Barreiro ML, Casanueva FF, Aguilar E, Dieguez C et al: Effects of KiSS-1 peptide, the natural ligand of GPR54, on follicle-stimulating hormone secretion in the rat. In: Endocrinology. vol. 146; 2005: 1689- 1697. 57. Navarro VM, Castellano JM, Fernández-Fernández R, Tovar S, Roa J, Mayen A, Nogueiras R, Vazquez MJ, Barreiro ML, Magni P et al: Characterization of the potent luteinizing hormone-releasing activity of KiSS-1 peptide, the natural ligand of GPR54. In: Endocrinology. vol. 146; 2005: 156-163. 58. Navarro VM, Fernández-Fernández R, Castellano JM, Roa J, Mayen A, Barreiro ML, Gaytan F, Aguilar E, Pinilla L, Dieguez C et al: Advanced vaginal opening and precocious activation of the reproductive axis by KiSS-1 peptide, the endogenous ligand of GPR54. In: J Physiol (Lond). vol. 561; 2004: 379-386.  40 59. Thompson EL, Patterson M, Murphy KG, Smith KL, Dhillo WS, Todd JF, Ghatei MA, Bloom SR: Central and peripheral administration of kisspeptin-10 stimulates the hypothalamic-pituitary-gonadal axis. J Neuroendocrinol 2004, 16(10):850-858. 60. Arreguin-Arevalo JA, Lents CA, Farmerie TA, Nett TM, Clay CM: KiSS-1 peptide induces release of LH by a direct effect on the hypothalamus of ovariectomized ewes. In: Anim Reprod Sci. vol. 101; 2007: 265-275. 61. Plant TM: The role of KiSS-1 in the regulation of puberty in higher primates. Eur J Endocrinol 2006, 155 Suppl 1:S11-16. 62. Shahab M, Mastronardi C, Seminara SB, Crowley WF, Ojeda SR, Plant TM: Increased hypothalamic GPR54 signalling: a potential mechanism for initiation of puberty in primates. In: Proc Natl Acad Sci USA. vol. 102; 2005: 2129-2134. 63. Dungan HM, Gottsch ML, Zeng H, Gragerov A, Bergmann JE, Vassilatis DK, Clifton DK, Steiner RA: The role of kisspeptin-GPR54 signalling in the tonic regulation and surge release of gonadotropin-releasing hormone/luteinizing hormone. In: J Neurosci. vol. 27; 2007: 12088- 12095. 64. Lapatto R, Pallais JC, Zhang D, Chan YM, Mahan A, Cerrato F, Le WW, Hoffman GE, Seminara SB: Kiss1-/- mice exhibit more variable hypogonadism than Gpr54-/- mice. In: Endocrinology. vol. 148; 2007: 4927-4936. 65. Han SK, Gottsch ML, Lee KJ, Popa SM, Smith JT, Jakawich SK, Clifton DK, Steiner RA, Herbison AE: Activation of gonadotropin-releasing hormone neurons by kisspeptin as a neuroendocrine switch for the onset of puberty. In: J Neurosci. vol. 25; 2005: 11349-11356. 66. Pielecka-Fortuna J, Chu Z, Moenter SM: Kisspeptin acts directly and indirectly to increase gonadotropin-releasing hormone neuron activity and its effects are modulated by estradiol. In: Endocrinology. vol. 149; 2008: 1979-1986. 67. Zhang C, Roepke TA, Kelly MJ, Rønnekleiv OK: Kisspeptin depolarizes gonadotropin-releasing hormone neurons through activation of TRPC-like cationic channels. In: J Neurosci. vol. 28; 2008: 4423-4434. 68. d'Anglemont de Tassigny X, Fagg LA, Carlton MB, Colledge WH: Kisspeptin can stimulate gonadotropin-releasing hormone (GnRH) release by a direct action at GnRH nerve terminals. In: Endocrinology. vol. 149; 2008: 3926-3932.  41 69. Keen KL, Wegner FH, Bloom SR, Ghatei MA, Terasawa E: An increase in kisspeptin-54 release occurs with the pubertal increase in luteinizing hormone-releasing hormone-1 release in the stalk-median eminence of female rhesus monkeys in vivo. In: Endocrinology. vol. 149; 2008: 4151-4157. 70. Gutiérrez-Pascual E, Martínez-Fuentes AJ, Pinilla L, Tena-Sempere M, Malagón MM, Castaño JP: Direct pituitary effects of kisspeptin: activation of gonadotrophs and somatotrophs and stimulation of luteinising hormone and growth hormone secretion. In: J Neuroendocrinol. vol. 19; 2007: 521-530. 71. Smith JT, Rao A, Pereira A, Caraty A, Millar RP, Clarke IJ: Kisspeptin is present in ovine hypophysial portal blood but does not increase during the preovulatory luteinizing hormone surge: evidence that gonadotropes are not direct targets of kisspeptin in vivo. In: Endocrinology. vol. 149; 2008: 1951-1959. 72. Clarkson J, d'Anglemont de Tassigny X, Moreno AS, Colledge WH, Herbison AE: Kisspeptin-GPR54 signalling is essential for preovulatory gonadotropin-releasing hormone neuron activation and the luteinizing hormone surge. In: J Neurosci. vol. 28; 2008: 8691-8697. 73. Smith JT, Cunningham MJ, Rissman EF, Clifton DK, Steiner RA: Regulation of Kiss1 gene expression in the brain of the female mouse. In: Endocrinology. vol. 146; 2005: 3686-3692. 74. Smith JT, Dungan HM, Stoll EA, Gottsch ML, Braun RE, Eacker SM, Clifton DK, Steiner RA: Differential regulation of KiSS-1 mRNA expression by sex steroids in the brain of the male mouse. In: Endocrinology. vol. 146; 2005: 2976-2984. 75. Clarkson J, Herbison AE: Postnatal development of kisspeptin neurons in mouse hypothalamus; sexual dimorphism and projections to gonadotropin-releasing hormone neurons. In: Endocrinology. vol. 147; 2006: 5817-5825. 76. Herbison AE, Theodosis DT: Localization of oestrogen receptors in preoptic neurons containing neurotensin but not tyrosine hydroxylase, cholecystokinin or luteinizing hormone-releasing hormone in the male and female rat. Neuroscience 1992, 50(2):283- 298. 77. Smith JT, Coolen LM, Kriegsfeld LJ, Sari IP, Jaafarzadehshirazi MR, Maltby M, Bateman K, Goodman RL, Tilbrook AJ, Ubuka T et al: Variation in kisspeptin and gonadotropin-inhibitory hormone expression and terminal connections to GnRH neurons in the brain: a novel medium for seasonal breeding in the sheep. In: Endocrinology. 2008.  42 78. Navarro VM, Castellano JM, Fernández-Fernández R, Barreiro ML, Roa J, Sánchez-Criado JE, Aguilar E, Dieguez C, Pinilla L, Tena-Sempere M: Developmental and hormonally regulated messenger ribonucleic acid expression of KiSS-1 and its putative receptor, GPR54, in rat hypothalamus and potent luteinizing hormone-releasing activity of KiSS-1 peptide. In: Endocrinology. vol. 145; 2004: 4565-4574. 79. Kinoshita M, Tsukamura H, Adachi S, Matsui H, Uenoyama Y, Iwata K, Yamada S, Inoue K, Ohtaki T, Matsumoto H et al: Involvement of central metastin in the regulation of preovulatory luteinizing hormone surge and estrous cyclicity in female rats. Endocrinology 2005, 146(10):4431- 4436. 80. Adachi S, Yamada S, Takatsu Y, Matsui H, Kinoshita M, Takase K, Sugiura H, Ohtaki T, Matsumoto H, Uenoyama Y et al: Involvement of anteroventral periventricular metastin/kisspeptin neurons in estrogen positive feedback action on luteinizing hormone release in female rats. In: J Reprod Dev. vol. 53; 2007: 367-378. 81. Kauffman AS, Gottsch ML, Roa J, Byquist AC, Crown A, Clifton DK, Hoffman GE, Steiner RA, Tena-Sempere M: Sexual differentiation of Kiss1 gene expression in the brain of the rat. In: Endocrinology. vol. 148; 2007: 1774-1783. 82. Roa J, Vigo E, Castellano JM, Gaytan F, Navarro VM, Aguilar E, Dijcks FA, Ederveen AG, Pinilla L, van Noort PI et al: Opposite roles of estrogen receptor (ER)-alpha and ERbeta in the modulation of luteinizing hormone responses to kisspeptin in the female rat: implications for the generation of the preovulatory surge. In: Endocrinology. vol. 149; 2008: 1627-1637. 83. Roa J, Vigo E, Castellano JM, Gaytan F, García-Galiano D, Navarro VM, Aguilar E, Dijcks FA, Ederveen AG, Pinilla L et al: Follicle-Stimulating Hormone Responses to Kisspeptin in the Female Rat at the Preovulatory Period: Modulation by Estrogen and Progesterone Receptors. In: Endocrinology. 2008. 84. Seminara SB, Dipietro MJ, Ramaswamy S, Crowley WF, Jr., Plant TM: Continuous human metastin 45-54 infusion desensitizes G protein- coupled receptor 54-induced gonadotropin-releasing hormone release monitored indirectly in the juvenile male Rhesus monkey (Macaca mulatta): a finding with therapeutic implications. Endocrinology 2006, 147(5):2122-2126. 85. Ramaswamy S, Guerriero KA, Gibbs RB, Plant TM: Structural Interactions Between Kisspeptin and GnRH Neurons in the Mediobasal Hypothalamus of the Male Rhesus Monkey (Macaca  43 Mulatta) as Revealed by Double Immunofluorescence and Confocal Microscopy. In: Endocrinology. 2008. 86. Shibata M, Friedman RL, Ramaswamy S, Plant TM: Evidence that down regulation of hypothalamic KiSS-1 expression is involved in the negative feedback action of testosterone to regulate luteinising hormone secretion in the adult male rhesus monkey (Macaca mulatta). In: J Neuroendocrinol. vol. 19; 2007: 432-438. 87. Kim W, Jessen HM, Auger AP, Terasawa E: Postmenopausal increase in KiSS-1, GPR54, and luteinizing hormone releasing hormone (LHRH-1) mRNA in the basal hypothalamus of female rhesus monkeys. In: Peptides. 2008. 88. Rometo AM, Krajewski SJ, Voytko ML, Rance NE: Hypertrophy and increased kisspeptin gene expression in the hypothalamic infundibular nucleus of postmenopausal women and ovariectomized monkeys. In: J Clin Endocrinol Metab. vol. 92; 2007: 2744-2750. 89. Rance NE, Uswandi SV: Gonadotropin-releasing hormone gene expression is increased in the medial basal hypothalamus of postmenopausal women. J Clin Endocrinol Metab 1996, 81(10):3540- 3546. 90. Rance NE, Young WS, 3rd: Hypertrophy and increased gene expression of neurons containing neurokinin-B and substance-P messenger ribonucleic acids in the hypothalami of postmenopausal women. Endocrinology 1991, 128(5):2239-2247. 91. Rance NE: Menopause and the human hypothalamus: evidence for the role of kisspeptin/neurokinin B neurons in the regulation of estrogen negative feedback. Peptides 2009, 30(1):111-122. 92. Kauffman AS, Clifton DK, Steiner RA: Emerging ideas about kisspeptin- GPR54 signalling in the neuroendocrine regulation of reproduction. Trends Neurosci 2007, 30(10):504-511. 93. Popa SM, Clifton DK, Steiner RA: The role of kisspeptins and GPR54 in the neuroendocrine regulation of reproduction. Annu Rev Physiol 2008, 70:213-238. 94. Smith JT: Sex steroid control of hypothalamic Kiss1 expression in sheep and rodents: comparative aspects. Peptides 2009, 30(1):94-102. 95. de Roux N, Genin E, Carel JC, Matsuda F, Chaussain JL, Milgrom E: Hypogonadotropic hypogonadism due to loss of function of the KiSS1-derived peptide receptor GPR54. Proc Natl Acad Sci U S A 2003, 100(19):10972-10976.  44 96. Semple RK, Achermann JC, Ellery J, Farooqi IS, Karet FE, Stanhope RG, O'Rahilly S, Aparicio SA: Two novel missense mutations in g protein- coupled receptor 54 in a patient with hypogonadotropic hypogonadism. J Clin Endocrinol Metab 2005, 90(3):1849-1855. 97. Lanfranco F, Gromoll J, von Eckardstein S, Herding EM, Nieschlag E, Simoni M: Role of sequence variations of the GnRH receptor and G protein-coupled receptor 54 gene in male idiopathic hypogonadotropic hypogonadism. Eur J Endocrinol 2005, 153(6):845- 852. 98. Tenenbaum-Rakover Y, Commenges-Ducos M, Iovane A, Aumas C, Admoni O, de Roux N: Neuroendocrine phenotype analysis in five patients with isolated hypogonadotropic hypogonadism due to a L102P inactivating mutation of GPR54. In: J Clin Endocrinol Metab. vol. 92; 2007: 1137-1144. 99. Teles MG, Bianco SD, Brito VN, Trarbach EB, Kuohung W, Xu S, Seminara SB, Mendonca BB, Kaiser UB, Latronico AC: A GPR54- activating mutation in a patient with central precocious puberty. In: N Engl J Med. vol. 358; 2008: 709-715. 100. Wacker JL, Feller DB, Tang XB, Defino MC, Namkung Y, Lyssand JS, Mhyre AJ, Tan X, Jensen JB, Hague C: Disease-causing Mutation in GPR54 Reveals the Importance of the Second Intracellular Loop for Class A G-protein-coupled Receptor Function. J Biol Chem 2008, 283(45):31068-31078. 101. d'Anglemont de Tassigny X, Fagg LA, Dixon JP, Day K, Leitch HG, Hendrick AG, Zahn D, Franceschini I, Caraty A, Carlton MB et al: Hypogonadotropic hypogonadism in mice lacking a functional Kiss1 gene. Proc Natl Acad Sci U S A 2007, 104(25):10714-10719. 102. Funes S, Hedrick JA, Vassileva G, Markowitz L, Abbondanzo S, Golovko A, Yang S, Monsma FJ, Gustafson EL: The KiSS-1 receptor GPR54 is essential for the development of the murine reproductive system. Biochem Biophys Res Commun 2003, 312(4):1357-1363. 103. Kauffman AS, Park JH, McPhie-Lalmansingh AA, Gottsch ML, Bodo C, Hohmann JG, Pavlova MN, Rohde AD, Clifton DK, Steiner RA et al: The kisspeptin receptor GPR54 is required for sexual differentiation of the brain and behavior. In: J Neurosci. vol. 27; 2007: 8826-8835. 104. Seminara SB: Metastin and its G protein-coupled receptor, GPR54: critical pathway modulating GnRH secretion. Front Neuroendocrinol 2005, 26(3-4):131-138.  45 105. Janneau JL, Maldonado-Estrada J, Tachdjian G, Miran I, Motte N, Saulnier P, Sabourin JC, Cote JF, Simon B, Frydman R et al: Transcriptional expression of genes involved in cell invasion and migration by normal and tumoral trophoblast cells. J Clin Endocrinol Metab 2002, 87(11):5336-5339. 106. Dhillo WS, Savage P, Murphy KG, Chaudhri OB, Patterson M, Nijher GM, Foggo VM, Dancey GS, Mitchell H, Seckl MJ et al: Plasma kisspeptin is raised in patients with gestational trophoblastic neoplasia and falls during treatment. Am J Physiol Endocrinol Metab 2006, 291(5):E878- 884. 107. Horikoshi Y, Matsumoto H, Takatsu Y, Ohtaki T, Kitada C, Usuki S, Fujino M: Dramatic elevation of plasma metastin concentrations in human pregnancy: metastin as a novel placenta-derived hormone in humans. J Clin Endocrinol Metab 2003, 88(2):914-919. 108. Torricelli M, Galleri L, Voltolini C, Biliotti G, Florio P, De Bonis M, Petraglia F: Changes of placental Kiss-1 mRNA expression and maternal/cord kisspeptin levels at preterm delivery. Reprod Sci 2008, 15(8):779-784. 109. Smets EM, Deurloo KL, Go AT, van Vugt JM, Blankenstein MA, Oudejans CB: Decreased plasma levels of metastin in early pregnancy are associated with small for gestational age neonates. In: Prenat Diagn. vol. 28; 2008: 299-303. 110. Thompson EL, Amber V, Stamp GW, Patterson M, Curtis AE, Cooke JH, Appleby GF, Dhillo WS, Ghatei MA, Bloom SR et al: Kisspeptin-54 at high doses acutely induces testicular degeneration in adult male rats via central mechanisms. Br J Pharmacol 2009, 156(4):609-625.    46 2 GPR54 AND KISS1 EXPRESSION IN CLINICAL CASES: ASSESSMENT OF PROSTATE, TESTICULAR, BREAST, AND OVARIAN CANCER TISSUE MICROARRAYS 1         Objective: To analyze GPR54 and kisspeptin by immunohistochemistry in a variety of cancers and determine any prognostic significance using linked clinical outcome data when available.         1  A version of this chapter will be submitted for publication. Prentice LM, Leung S, Köbel M, McKinney S, Goktepe O, Rajput A, Mehl E, Gilks CB, Huntsman DG, and Aparicio SAJ. GPR54 and KiSS1 expression in clinical cases: assessment of prostate, testicular, breast, and ovarian cancer tissue microarrays.  47 2.1 Introduction With the discovery of the KiSS1 gene as a metastasis suppressor, many researchers set out to explore KiSS1 expression in a multitude of cancers to determine if the kisspeptin (Kp) protein could be used as a prognostic biomarker. Similarly, expression of the Kp receptor, GPR54, was explored alongside Kp in several studies. The difficulty in achieving this aim was establishing specific antibodies for both Kp and GPR54. Therefore many of these studies utilized RNA in situ hybridization or real-time PCR (QPCR) as an alternative to immunohistochemical (IHC) analysis.  The normal physiological function of Kp and GPR54 is to regulate the hypothalamic-pituitary-gonadal axis, however both are expressed in tissues outside of the hypothalamus. KiSS1 is normally expressed at low levels in the testes, liver, pancreas, and small intestine and highly expressed in the placenta [1, 2]. GPR54 is also most highly expressed in the placenta but also has high expression in the pituitary and pancreas. GPR54 is expressed at lower levels in the testes, spleen, thymus, and lymph nodes [1-3].  To assess the prognostic significance of this ligand-receptor pair in clinical cases, KiSS1 and GPR54 expression has been examined in a variety of cancers. In almost all cases loss of Kp and/or GPR54 has been correlated with poor prognosis or advanced disease. This finding has been true for gastric, esophageal, bladder, melanoma, pancreatic, thyroid, renal and breast cancer [4- 11]. The one anomaly has been hepatocellular cancer where a more advanced  48 disease was associated with overexpression of KiSS1 and GPR54 as determined by QPCR [12].  Using immunohistochemistry and tissue microarray technology (TMA), we assessed Kp and GPR54 expression in cancers that had not been previously evaluated: ovarian, testicular, and prostate. We also assessed a large 438 case breast cancer TMA with clinically linked outcome data to assess prognostic significance. Fluorescent in situ hybridization was also employed to determine gene copy number as a possible mechanism for KiSS1 and GPR54 expression in the breast and ovarian cancer tissue microarrays.  2.2 Material and Methods 2.2.1 Tissue microarrays Breast cancer array The breast cancer array was constructed using formalin-fixed, paraffin embedded tissue blocks received from the Department of Pathology at Vancouver General Hospital during the period 1974-1995. Case selection and tissue microarray construction have been described previously [13-15]. Briefly, representative areas of carcinoma were selected and marked on the hematoxylin and eosin slides and corresponding tissue blocks for TMA construction. The TMAs were assembled using a tissue-arraying instrument (Beecher Instruments, Silver Springs, MD), with two 0.6 mm cores per case. Outcome data were available for all 438 patients, with median follow-up of 15.4 years (range 6.3-26.6 years). Ethical approval was obtained to perform this study from the Clinical Research  49 Ethics Board of the University of British Columbia. The clinicopathological characteristics of the cohort are listed in Table 2.1.  Prostate cancer array The prostate array consisted of 84 quadruplicate 0.6 mm cores obtained from formalin-fixed paraffin embedded tissue blocks as previously described [16]. The samples were obtained from radical prostatectomies from non-treated patients of varying Gleason grade as reviewed by a pathologist. The clinicopathological characteristics of the cohort are listed in Table 2.2.  Germ cell testis array Duplicate 0.6 mm cores from paraffin embedded germ cell tumours (33 seminomas, 23 pure embryonal carcinomas and 10 mixed embryonal carcinomas) obtained from the Brigham and Women’s Hospital were used to create the testicular TMA under the Harvard Medical School Institutional Review Board guidelines [17]. The clinicopathological characteristics for this cohort were unavailable.  Ovarian array This small ovarian tissue microarray consists of 49 ovarian cancer samples that were obtained from patients who underwent debulking surgery from January 2004 until September 2005 at Vancouver General Hospital and the British Columbia Cancer Agency. Patients treated with pre-operative chemotherapy  50 Table 2.1 Clinicopathological characteristics of the breast TMA  Parameter N Histology Invasive Ductal 372 Invasive Lobular 40 Node Negative 236 Positive 134 Grade 1 84 2 219 3 100 Biomarkers ER <1% 78 ER >=1% 287 Her2 neg 240 Her2 weak 69 Her2 strong  35 Tumour Size <=2cm 185 >2cm and <=5cm 186 >5cm 34 Age (years) Mean (SD) 60.3 (14.1)   51  Table 2.2 Clinicopathological characteristics of the prostate TMA  Parameter N Histology BPH 16 GL3 38 GL4 7 GL5 23 Clinical Stage T1 a, b, c 30 T2 a, b 9 T3 a, b 1 Pathological Stage T2 a, b 54 T3 a, b 10 Age (years) Median 66 Range (Min – Max) 46-89   52  were omitted [18]. TMA construction was similar to that described in the breast cancer array and ethical approval was obtained from the University of British Columbia Ethics Board. The clinicopathological characteristics of the cohort are listed in Table 2.3.  2.2.2 Manual kisspeptin immunohistochemistry For the breast cancer TMA, kisspeptin immunohistochemistry (IHC) was performed manually. Sections from formalin-fixed and paraffin-embedded tissues were deparaffinized with xylene and rehydrated with a graded series of alcohols. Wet heat-induced antigen retrieval was performed in a steamer for 20 min with a modified citrate buffer (pH 6.1, Dako, Mississauga, ON). Following antigen retrieval, sections were treated with 3% hydrogen peroxide (H2O2) in phosphate buffered saline (PBS) for 30 min to quench endogenous peroxidase activity. All of the aforementioned steps were followed by three washes with PBS for 5 min each. Slides were subsequently blocked for 30 min with serum-free protein block (Dako) and incubated overnight at 4°C with a polyclonal goat anti-KiSS-1 antibody (C-20, Santa Cruz Biotechnology, Santa Cruz, CA) diluted 1:400 in serum-free protein block. Kisspeptin immunoreactivity (IR) was detected with the CSA II biotin-free tyramide signal amplification system and 3,3’-diaminobenzidine chromogen solution (Dako). Specifically, rabbit anti-goat-horseradish peroxidase (HRP) was applied for 15 min followed by fluorescyl-tyramide amplification reagent for 15 min and anti-fluorescein-HRP for 15 min. All of the steps subsequent to the incubation with primary antibody were followed by three  53 Table 2.3 Clinicopathological characteristics of the ovarian TMA  Parameter N Histopathological Subtype Serous 40 Endometrioid 6 Clear Cell 4 Serous Borderline 1 Grade 1 4 2 7 3 39  Stage 1 7 2 7 3 30 4 7 Age (years) Mean (SD) 59.5 (12.2) Median 57.0   54  washes with tris-buffered saline containing 1% Tween (TBST) for 5 min each. Slides were counterstained with Harris hematoxylin (Sigma-Aldrich, Oakville, ON) and mounted in a xylene-based mounting medium.  Based on previously published data showing cell-type restriction of GPR54 and kisspeptins in different trophoblast layers of human placenta (Bilban 2004), less than 10-week old human placenta was used as a specificity control (courtesy of Vancouver Coastal Health archives), in conjunction with two blocking peptides (21 residues and 54 residues). Omission of the primary antibody was used as a negative control.  2.2.3 Automated GPR54 and kisspeptin immunohistochemistry All of the TMAs were stained using an automated system for both GPR54 and Kp IHC, except for the breast TMA that is described above. TMA 4 m sections were processed using a Ventana Discovery XT automated system (Ventana Medical Systems, Tucson, AZ) as per manufacturer’s protocol with proprietary reagents. After slides were baked at 60°C for 1 h, they were deparaffinized on the automated system with EZ Prep solution (Ventana). Heat-induced antigen retrieval method was used in Cell Conditioning solution (CC1- Tris based EDTA buffer, pH 8.0, Ventana).  The polyclonal rabbit GPR54 antibody was obtained from MBL International Corporation (Woburn, MA) specific for the N-terminal extracellular domain (catalogue number LS-A1929) and used with heat at a 1:25 concentration in Ventana antibody diluent. The polyclonal goat KiSS1 antibody from Santa Cruz (Santa Cuz, CA) was incubated for 2 h without heat at 1:10 dilution. For GPR54, the Ventana Universal Secondary Antibody was used for 32  55 min at 37°C, while a rabbit anti-goat (Jackson ImmunoResearch, West Grove, PA) at 1:500 dilution was used for KiSS1.  The detection system used for GPR54 was the Ventana DABMap kit, while the Ultra detection system (Ventana) was used for KiSS1. All slides were counterstained with Hematoxylin and treated with a proprietary bluing agent (Ventana) and washes were conducted with the Ventana Reaction Buffer.  Dehydration steps and coverslip procedure were completed manually as per manufacturer’s recommendations. Specificity was determined by Western blot for GPR54 and by using less than 10-week old human placenta as a positive control and omission of primary antibody as a negative control for both antibodies.  2.2.4 IHC scoring Kp and GPR54 IHC scoring was assessed by a pathologist (Martin Köbel or Ashish Rajput) for all tissues except Kp in the ovarian carcinomas. For all TMAs, GPR54 was scored as 0 for tissues with fewer than 5% of staining, +1 for tissues that had at least 5% of tumour cells with dark staining, and +2 for tissues that had greater than 75% of tumour cells with dark staining. For all the TMAs except the small ovarian cancer array, Kp was scored at 0 for completely negative tissues, +1 for any detectable staining, and +2 for intense signal. For the ovarian cancer array, Kp was scored solely by the author (Leah Prentice) without the benefit of a trained pathologist and was considered 0 for completely negative tissues, +1 for very weak staining, +2 for moderate staining, and +3 for intense staining.   56 2.2.5 Photomicrographs The TMAs were digitally scanned with a BLISS (Bacus Laboratories Inc., Slide Scanner) automated system (Bacus Laboratories, Lombard, IL) as previously described [19]. These images are publically available on our webslide server: http://www.gpecimage.ubc.ca/tma/web/viewer.php.  2.2.6 Fluorescent in situ hybridization (FISH) Six mm thick sections of the TMAs were treated for fluoresecent in situ hybridization (FISH) as described elsewhere [20, 21]. Locus specific FISH analysis for KiSS1 and GPR54 were performed using the BACs CTD-3195E18, CTD-2582H10, and CTD3009K5 for GPR54 and RP11-673P13, CTD-2145H2, and RP11-203F10 for KiSS1 (BACPAC Resources Centre, Children’s Hospital Oakland Research Institute). All BACs were directly labeled with Spectrum Green using a Nick translation kit (Vysis, IL, USA), and validated for specificity using normal metaphases from human blood cells. A centromeric probe for chromosome 1 and a telomeric probe control for 19q both labeled in spectrum orange were used as controls for aneusomy for KiSS1 and GPR54 respectively (Vysis, IL, USA). FISH was performed using Vysis reagents according to the manufacturer’s protocols (Vysis, IL, USA) and slides were counterstained with 4,6-diamidino 2-phenylindole (DAPI). FISH signals and patterns were identified on a Zeiss Axioplan epifluorescent microscope and were scored either manually (oil immersion 100X) or using Metasystems Metafer software (MetaSystems Group, Inc. Belmont, MA) and enumerated in approximately 40 morphologically  57 intact and non-overlapping nuclei. Amplification was defined as an amplification ratio of 1.5 or greater, and loss was defined as a ratio of 0.6 or less.  2.2.7 Reverse transcription The ovarian TMA had matched RNA available from Press et al. [18] for most of the cases. One microgram of RNA was DNAse treated prior to reverse transcription to eliminate any contaminating genomic DNA from the sample. Samples were incubated at RT with DNAse I (Invitrogen, Carlsbad, CA) for 15 min before the addition of 25 mM EDTA (Invitrogen, Carlsbad, CA) and heated to 65°C for 10 min to stop the reaction as per manufacturer’s recommendation. Samples were then immediately put on ice to cool. Random hexamers at 250 ng/ul (Invitrogen, Carlsbad, CA), 10 μM dNTPs (Invitrogen, Carlsbad, CA), 400 U MMLV-Reverse transcriptase enzyme (Invitrogen, Carlsbad, CA), 0.1 M DDT (Invitrogen, Carlsbad, CA), with the accompanying reverse transcriptase buffer (250 mM Tris-HCI pH 8.3, 375 mM KCl, 15 mM MgCl2), 8 U RNase OUT enzyme, and water were added to the DNAse treated samples. Samples were incubated at 23°C for 10 min then incubated at 37°C for 1 h, and the reaction was stopped by a 10 min 65°C incubation step.  2.2.8 Quantitative real-time PCR (QPCR) The Applied Biosystems (ABI) 7900HT Fast Real-Time System was used to amplify and detect KiSS1 and GPR54 mRNA transcripts (Applied Biosystems, Foster City, CA). Gene specific probe and primer sets were ordered as 20x target  58 assays from ABI specifically covering exon boundaries for human KiSS1 (Hs00158486_m1) and GPR54 (Hs00261399_m1). The 18s rRNA subunit (Hs99999901_s1) expression levels at 1:250 dilution were used as an endogenous loading control before comparative normalization. Using RNA generously donated by Dr. Lindsay Brown, KiSS1 expression was normalized to RNA from IOSE epithelial ovarian cells using relative quantification. GPR54 expression was normalized to RNA from MDA-MB-231 cells, as IOSE cells did not express GPR54.  2.2.9 Statistics For the breast TMA Kp and GPR54 correlations with clinicopathological markers and survival, were assessed using statistical software SPSS 16.0. For boxplots demonstrating the GPR54 and KiSS1 gene copy number and IHC relationship, R 2.8 software was used. For the small ovarian TMA, Kp and GPR54 continuous variables (QPCR results) were compared by linear regression and compared to discrete variables (FISH ratios, IHC scores) using Fischer's ANOVA (analysis of variation). Discrete variables compared to discrete variables were analyzed by Pearson's chi-square.   59 2.3 Results 2.3.1 GPR54 is more highly expressed in the more benign subtype of prostate and testicular cancers We assessed a prostate tissue microarray for Kp and GPR54 expression by immunohistochemistry. Contained within the prostate array were cases of benign prostatic hyperplasia (BPH) as well as cases that ranged in Gleason score from GL3-GL5. Of these cases, GPR54 was most frequently and most highly expressed in the BPH (85% of cases had an IHC score of +2). There was no discernable difference between the remaining Gleason grades, nor was there any detectable differences between the kisspeptin expressing cases (Figure 2.1 A). The lack of any obvious expression pattern within subtype for Kp could also be due to the lack of Kp positivity in this tissue. Of 72 scorable cases, only 6 had any detectable Kp (8%).  The testicular array consisted of germ cell tumours, either seminomas, or nonseminomas (embryonal mixed or embryonal pure). Of these cancers, the seminomas have a better prognosis and 32/33 (97%) cases had high GPR54 expression (IHC score of +2). KiSS1 was also most highly and most frequently expressed in this subtype with an average IHC score of +1 (Fig 2.1 B). Overall, Kp and GPR54 were more frequently and highly expressed in the testicular cancers rather than the prostate cancers.   60   Figure 2.1 GPR54 and Kp expression in male cancers Frequency of expression is measured on the left y-axis as percent of positive cases represented by bars, while strength of expression is the average IHC score within a particular subtype measured by the right y-axis and is represented by squares. A) Prostate cancer TMA. B) Germ cell TMA (GPR54 in red on left side; Kp in blue on right side). Error bars for the average IHC scores represents the standard error across all samples within a particular subtype. E=embryonal.  61 2.3.2 Loss of GPR54 in breast cancer is a poor prognostic marker in node positive patients. To assess whether Kp and GPR54 expression have prognostic significance, a breast cancer TMA with linked clinical outcome data was used. Both Kp and GPR54 were grouped individually into negative and positive cases based on their IHC expression. As GPR54 is normally expressed in breast tissue (Fig 2.2 A), the negative expression group consisted of cases with an IHC score of 0 and was considered as GPR54 loss. The GPR54 positive group consisted of the remaining +2 and +1 IHC cases. Kp is not ordinarily expressed in breast tissue (Figure 2.2 A), and those with an IHC score of +2 were considered as positive. The remaining 0 and +1 cases were grouped and classified as Kp negative. Loss of GPR54 was significantly associated with poor prognosis in node positive patients in overall and disease specific survival (Figure 2.2 B), but this did not maintain significance within multivariable analysis against age at diagnosis and Her2 positivity (Table 2.4). Though Kp expression alone did not correlate with survival, breast cancer cases expressing both Kp and GPR54 have a significantly more favourable prognosis when compared to those that had both loss of Kp and GPR54 in disease specific survival (Figure 2.2 C).  Kp and GPR54 expression did not significantly correlate with any other clinicopathological markers such as estrogen receptor status, or Her2 expression after a correction for multiple comparisons (Tables 2.5 and 2.6 respectively). There was a slight positive correlation between the highest Kp and GPR54 expressors (+2) (Kendall’s tau-b= 0.199, p=0.0013, Table 2.5).   62  Figure 2.2 Kp and GPR54 expression in breast cancer A) Representative micrographs of Kp (left) and GPR54 (right) expression in normal human breast tissue. B) Kaplan-Meier survival plots with cumulative survival on the y-axis and time in years on the x-axis for disease specific (DSS) and overall survival (OS). C) Kaplan-Meir survival plot including both Kp and GPR54.  63 Table 2.4 Multivariable analysis in node positive breast cancer patients  Source Hazard Ratio 95% CI L 95% CI U p-value GPR54 0.828 0.291 2.352 0.723 Grade 0.941 0.545 1.625 0.827 ER 0.973 0.370 2.557 0.955 Age at Dx 1.030 1.008 1.053 0.007 Tumour Size 1.089 0.896 1.324 0.390 Her2 9.627 2.940 31.524 0.000     Table 2.5 Kp IHC correlation with clinicopathological markers  Biomarker  Kendall's tau-b Raw p-value GPR54 [0,1] vs. [2]  0.199 0.001* KI67 cut off at 13.5%  -0.136 0.028 Grade [1,2] vs.[3]  -0.117 0.051 Her2 [0,1] vs. [2]  -0.120 0.073 PR cut off at 1%  0.081 0.240 BCL2 [0] vs. [1,2]  0.076 0.241 ER cut off at 1%  0.060 0.408 Age cut off at 50 yrs  0.051 0.456 CK5/6 [0] vs. [1,2]  -0.041 0.578 KISS1 FISH cut off at 1.5  -0.053 0.592 EGFR [0] vs. [1,2]  -0.031 0.685 Nodal status  -0.022 0.777 Tumor size cut off at 2 cm  0.012 0.894 *Maintains significance with consideration for multiple comparisons.              64 Table 2.6 GPR54 IHC correlations with clinicopathological markers  Biomarker  Kendall's tau-b Raw p-value Tumor size cut off at 2 cm  -0.116 0.036 PR cut off at 1%  -0.093 0.165 KI67 cut off at 13.5%  0.076 0.207 EGFR [0] vs. [1,2]  0.070 0.331 Grade [1,2] vs.[3]  0.046 0.492 Nodal status  0.031 0.492 BCL2 [0] vs. [1,2]  -0.043 0.497 Her2 [0,1] vs. [2]  -0.025 0.718 ER cut off at 1%  0.009 0.809 Age cut off at 50 yrs  -0.028 0.820 CK5/6 [0] vs. [1,2]  0.003 1.000 GPR54 FISH cut off at 1.4  0.062 1.000   65 2.3.3 GPR54 and Kp expression is not mediated through gene copy number in breast cancer To assess gene copy number as a possible mechanism for either increase or loss of Kp and/or GPR54 expression, a fluorescent in situ hybridization (FISH) assay was established and applied to the breast cancer TMA (Figure 2.3 A). Gene copy number is compared to a reference probe, in this instance either centromere 1 for KiSS1 or telomere 19q for GPR54, to take into account any abnormal gene copy number that may be due to chromosomal aneuploidy. For GPR54 analysis there were 191 breast cancer cases with scorable results. Of these, 27 (14%) had loss of GPR54 using a ratio of 0.6 or less, and 4 had gain of GPR54 using a ratio of 1.5 or greater. For the KiSS1 locus, 29 of 233 scorable cases were amplified (12.4%) and one had loss of gene copy number, using the same ratio parameters described for GPR54. KiSS1 amplification ratio did not correlate with Kp expression (Spearman's =-.05, p=0.529), while surprisingly GPR54 gene copy number had a slight negative correlation with the IHC results (=-.195, p=0.019; Figure 2.3 B). For GPR54, gene copy number was used rather than a ratio of loss to take into account any low ratios arising from abnormal chromosomal gain.  2.3.4 GPR54 and Kp expression is not mediated through gene copy number in ovarian cancer Using a small ovarian cancer array, GPR54 and KiSS1 gene copy number was determined by FISH, mRNA expression was determined by real-time PCR, and protein expression was determined by IHC (Tables 2.7 and 2.8 respectively).  66 Figure 2.3 GPR54 and KiSS1 gene copy number in breast cancer A) Fluorescent in situ hybridization (FISH) was used to determine gene copy number for GPR54 (left) and KiSS1 (right). Top panel shows gene expression in normal metaphase demonstrating probe specificity; middle panel shows normal gene copy number in tumours. Bottom panel shows loss for GPR54 (green) as compared to 19q telomeric control (orange), while KiSS1 (green) is amplified compared to centromere 1 control (orange). Locations of the probes are shown on the corresponding idiograms of the chromosome being studied. B) Boxplots comparing gene copy number for GPR54 with IHC score (left) or gene ratio for KiSS1 with IHC score (right).   67      68 Table 2.7 GPR54 gene ratio and expression in ovarian cancer Tumor  QPCR FISH Ratio IHC  Histopathology 198 56.16 x x Clear cell 213 0.66 x x Clear cell 219 8.65 x x Clear cell 392 1.04 x 2 Clear cell 242 1.12 1.0 2 Endometrioid 281 9.43 x 2 Endometrioid 334 3.28 1.0 2 Endometrioid 156 0.91 1.1 0 Endometrioid 343 x x x Endometrioid 172 0.45 1.1 2 Mixed carcinoma 186 0.46 1.1 2 Mixed carcinoma 240 2.73 0.8 2 Mixed carcinoma 327 0.98 x 1 Mixed carcinoma 280 0.21 1.5 0 Poorly differentiated 336 0.04 1.0 2 Poorly differentiated 161 0.96 x 2 Poorly differentiated 201 3.55 x 1 Poorly differentiated 208 0.37 x x Poorly differentiated 273 7.09 x 0 Poorly differentiated 305 0.69 x 2 Poorly differentiated 363 0.06 0.4 2 Primary peritoneal 221 7.27 1.1 2 Serous 324 1.17 x 2 Serous 163 2.46 0.9 1 Serous 178 1.56 1.0 1 Serous 195 4.47 2.0 1 Serous 212 1.38 1.2 2 Serous 217 0.79 1.1 0 Serous 223 2.22 1.1 2 Serous 229 0.41 0.6 0 Serous 236 0.10 x x Serous 239 1.82 1.1 2 Serous 254 0.04 x 1 Serous 283 1.99 x 2 Serous 293 0.39 0.5 2 Serous 297 0.91 x x Serous 309 0.67 x x Serous 319 1.36 x 1 Serous 330 1.46 0.6 2 Serous 332 0.62 1.0 2 Serous 344 1.37 1.1 2 Serous 345 0.92 x 2 Serous 366 0.31 x 2 Serous 372 6.00 1.1 0 Serous 379 0.96 x 2 Serous 384 6.50 x x Serous 388 13.68 1.0 1 Serous 394 x 0.6 2 Serous 329 0.03 0.9 2 Serous  69 Table 2.8 KiSS1 gene ratio and expression in ovarian cancer Tumor  QPCR FISH Ratio IHC Histopathology 198 0.05 0.7 3 Clear cell 213 0.59 x 3 Clear cell 219 1.66 x 3 Clear cell 392 1.16 1.6 3 Clear cell 242 1.77 x 1 Endometrioid 281 9.27 1.1 2 Endometrioid 334 0.33 1.0 1 Endometrioid 156 7.44 x 1 Endometrioid 343 0.21 x 0 Endometrioid 172 7.20 1.7 3 Mixed carcinoma 186 x 1.0 2 Mixed carcinoma 240 1.20 1.1 2 Mixed carcinoma 327 1.36 1.1 0 Mixed carcinoma 280 2.25 0.9 0 Poorly differentiated 336 2.62 0.8 1 Poorly differentiated 161 5.12 x 1 Poorly differentiated 201 2.68 x 0 Poorly differentiated 208 0.04 x 1 Poorly differentiated 273 3.27 x 0 Poorly differentiated 305 1.69 1.1 2 Poorly differentiated 363 0.28 0.7 0 Primary peritoneal 221 0.92 1.2 0 Serous 324 0.75 0.9 0 Serous 163 1.42 1.4 0 Serous 178 0.94 0.5 0 Serous 195 x 0.9 0 Serous 212 0.30 1.5 0 Serous 217 3.02 1.0 0 Serous 223 0.77 0.9 1 Serous 229 3.01 0.8 0 Serous 236 0.34 x 0 Serous 239 57.18 1.4 0 Serous 254 0.33 0.7 0 Serous 283 2.36 1.1 0 Serous 293 0.98 1.1 1 Serous 297 0.02 x 0 Serous 309 0.17 x 2 Serous 319 0.05 1.1 1 Serous 329 0.27 1.1 1 Serous 330 3.75 1.0 1 Serous 332 1.95 1.2 1 Serous 344 x 1.0 0 Serous 345 0.34 1.4 0 Serous 366 0.64 1.3 0 Serous 372 1.91 0.9 0 Serous 379 1.27 1.2 0 Serous 384 0.53 x 0 Serous 388 1.05 1.3 0 Serous 394 x 0.9 0 Serous  70 For GPR54, 5/26 cases had a FISH ratio of 0.6 or less, 14/47 had a QPCR fold change of 2 or greater, and 6/40 cases were negative for GPR54 protein (IHC score of 0). There was a slight yet significant positive correlation between FISH gene ratio and real-time PCR expression (r2=0.167, p=0.03822). However there was no significant correlation between either gene ratio and IHC or QPCR and IHC. GPR54 IHC was only resolved in 1 of 4 clear cell cases and did not appear to favour any particular subtype. For KiSS1, 3/36 cases showed gene amplification by FISH using a cut-off ratio of 1.5 or greater, 13/45 cases showed an expression fold change of 2 or greater for QPCR, and 22/49 cases showed Kp expression by IHC (IHC scores +1, +2, and +3 grouped). None of these parameters significantly correlated with one another, most specifically gene ratio with IHC. Of note, all four cases of the ovarian cancer clear cell subtype had the highest Kp IHC scores.  2.4 Conclusions We have demonstrated for the first time that GPR54 is more highly expressed and at a greater frequency in benign prostate hyperplasia as compared to malignant disease. Benign prostatic hyperplasia results in the enlargement of the prostate that leads to urinary tract problems but is not believed to be a premalignant lesion [22]. Kp and GPR54 protein expression in normal prostate tissue is currently unknown, and therefore it cannot be determined if GPR54 expression is gained in benign prostatic hyperplasia or lost in prostate cancer. However, high GPR54 expression in benign disease while having lower  71 expression in malignant disease is consistent with the hypothesis that gain of expression is correlated with a more favourable phenotype. Comparatively Kp expression is a rare event in prostate disease.  Germ-cell cancers are the most common type of cancer in young men and are separated into seminomas and nonseminomas. Seminomas are generally confined to the testis and not considered as high risk. Conversely, half of the patients that are diagnosed with nonseminomas have metastatic spread [23]. Almost all the seminomas examined in this TMA had the highest GPR54 expression, only one case had moderate GPR54 expression. Kp also had the highest expression in seminomas though the results were not as striking as for GPR54. Part of assessing testicular germ cell cancers for risk and confirming diagnosis is the association with serum markers. Nonseminomas may express - fetoprotein (yolk sac tumours) or human chorionic gonadotropin (hCG; choriocarcinomas) and lactate dehydrogenase (LDH) though the latter is not specific to germ cell tumours. Seminomas have been shown to express hCG in plasma in 20% of patients. In general the higher the concentration of the serum marker the more advanced the cancer and the higher the stage [23]. hCG is also highly expressed in gestational trophoblast neoplasia (GTN), a malignant tumour of placental origin. This is not surprising as hCG is secreted from normal healthy placenta, as is Kp [24, 25]. It was recently discovered that Kp is also elevated in GTN and decreases during treatment until it reaches the normal concentrations once the tumour is completely removed [24]. Due to the similarity in Kp and hCG  72 expression in GTN, this raises the possibility that Kp may also be secreted from testicular germ cell tumours that secrete hCG. As Kp is more highly expressed in the seminomas, and least in the embryonal pure nonseminomas, it may be used as a plasma marker to distinguish between subtype of germ cell tumours and aid in determining treatment. This is an area of research yet to be assessed. In breast cancer, loss of GPR54 correlates with poor prognosis in node positive patients. Node positivity is associated with increased risk for metastasis and metastasis is the main cause of death in breast cancer patients [26]. This correlation was significant in disease specific and overall survival but was not an independent marker when compared to other well established risk factors such as Her2 positivity. Due to this outcome and that Kp was insignificant in all analysis make this ligand-receptor pair a poor consideration as biomarkers in breast cancer. However, these findings support our hypothesis of GPR54 expression being associated with favourable outcome.  To elucidate a possible mechanism for Kp and GPR54 protein expression, we assessed gene copy number and RNA expression of this ligand-receptor pair. We were unable to find any significant correlation between DNA, RNA, and protein, however this is not the first report of unmatched expression. Specifically, Kiss1 expression as determined by RNA in situ hybridization was localized in the arucate nucleus (ARC), the anteroventral periventricular nucleus (AVPV) and the periventricular nucleus (PeN) of the female mouse hypothalamus [27, 28], but only located in the AVPV and ARC by IHC [28]. Furthermore, Kiss1 mRNA was  73 much lower and sometimes completely absent in the brain of rats as compared to Kp protein as determined by IHC [29]. Kp is a secreted protein and tissues with high RNA expression may be providing Kp for neighbouring tissues or for endocrine signalling. Depending on the speed at which Kp is manufactured and released from the cell, Kp protein may not be measureable in the Kp donating tissues and this may account for differences between RNA and protein expression. Alternatively, Kp has an extremely short half-life of 27.6 ± 1.1 min [30] in plasma, which may reflect a short half-life within tissues. Kp may be more rapidly degraded in formalin-fixed, paraffin embedded tissues used in TMAs as compared to frozen tissues that are used to provide RNA for the analysis. For GPR54, a rapid change in gene expression may not be reflected immediately in protein concentration for a seven-transmembrane protein. The half-life of GPR54 has yet to be determined, but transmembrane proteins may be internalized and recycled back to the surface without actual degradation of protein thus increasing the half-life significantly [31]. Therefore it follows that there may be low levels of GPR54 mRNA within tissues that have high levels of GPR54 protein. Further studies are required to definitively account for differences in RNA and protein expression.  We have also determined that gene copy number does not account for gain or loss of either Kp or GPR54 expression in breast and ovarian cancer. Gene copy number can account for increased protein expression as has been seen in HER2/ErbB2. HER2 is overexpressed in 15-25% of ductal breast cancers  74 and correlates with poor prognosis [32]. Clinically ErbB2 expression is detected by IHC and followed up by FISH when the initial results are unclear. If FISH results determine that Her2 is amplified, then treatment for the patient will follow the regiment of Herceptin treatment to block the ErbB2 receptor. However, gene copy number is not always responsible for protein expression as in the case of ER [33].  This is the first time GPR54 and Kp expression has been determined by IHC in testicular, prostate, and ovarian cancer. Although previously assessed in breast cancer, this is the first report of expression determined in a large cohort with linked clinical outcome data. In all of these diseases, expression correlated with the characteristics linked to a more favourable prognosis. This research supports the current literature that expression of this ligand-receptor pair is a favourable prognostic marker. However, further studies are required before either Kp or GPR54 could be considered as reliable cancer biomarkers.   75 2.5 References 1. Muir AI, Chamberlain L, Elshourbagy NA, Michalovich D, Moore DJ, Calamari A, Szekeres PG, Sarau HM, Chambers JK, Murdock P et al: AXOR12, a novel human G protein-coupled receptor, activated by the peptide KiSS-1. J Biol Chem 2001, 276(31):28969-28975. 2. Ohtaki T, Shintani Y, Honda S, Matsumoto H, Hori A, Kanehashi K, Terao Y, Kumano S, Takatsu Y, Masuda Y et al: Metastasis suppressor gene KiSS-1 encodes peptide ligand of a G-protein-coupled receptor. Nature 2001, 411(6837):613-617. 3. Kotani M, Detheux M, Vandenbogaerde A, Communi D, Vanderwinden JM, Le Poul E, Brézillon S, Tyldesley R, Suarez-Huerta N, Vandeput F et al: The metastasis suppressor gene KiSS-1 encodes kisspeptins, the natural ligands of the orphan G protein-coupled receptor GPR54. In: J Biol Chem. vol. 276; 2001: 34631-34636. 4. Dhar DK, Naora H, Kubota H, Maruyama R, Yoshimura H, Tonomoto Y, Tachibana M, Ono T, Otani H, Nagasue N: Downregulation of KiSS-1 expression is responsible for tumor invasion and worse prognosis in gastric carcinoma. Int J Cancer 2004, 111(6):868-872. 5. Ikeguchi M, Yamaguchi K, Kaibara N: Clinical significance of the loss of KiSS-1 and orphan G-protein-coupled receptor (hOT7T175) gene expression in esophageal squamous cell carcinoma. Clin Cancer Res 2004, 10(4):1379-1383. 6. Masui T, Doi R, Mori T, Toyoda E, Koizumi M, Kami K, Ito D, Peiper SC, Broach JR, Oishi S et al: Metastin and its variant forms suppress migration of pancreatic cancer cells. Biochem Biophys Res Commun 2004, 315(1):85-92. 7. Ringel MD, Hardy E, Bernet VJ, Burch HB, Schuppert F, Burman KD, Saji M: Metastin receptor is overexpressed in papillary thyroid cancer and activates MAP kinase in thyroid cancer cells. J Clin Endocrinol Metab 2002, 87(5):2399. 8. Sanchez-Carbayo M, Capodieci P, Cordon-Cardo C: Tumor suppressor role of KiSS-1 in bladder cancer: loss of KiSS-1 expression is associated with bladder cancer progression and clinical outcome. Am J Pathol 2003, 162(2):609-617. 9. Shirasaki F, Takata M, Hatta N, Takehara K: Loss of expression of the metastasis suppressor gene KiSS1 during melanoma progression  76 and its association with LOH of chromosome 6q16.3-q23. Cancer Res 2001, 61(20):7422-7425. 10. Shoji S, Tang XY, Umemura S, Itoh J, Takekoshi S, Shima M, Usui Y, Nagata Y, Uchida T, Osamura RY et al: Metastin Inhibits Migration and Invasion of Renal Cell Carcinoma with Overexpression of Metastin Receptor. In: Eur Urol. 2008. 11. Stark AM, Tongers K, Maass N, Mehdorn HM, Held-Feindt J: Reduced metastasis-suppressor gene mRNA-expression in breast cancer brain metastases. J Cancer Res Clin Oncol 2005, 131(3):191-198. 12. Ikeguchi M, Hirooka Y, Kaibara N: Quantitative reverse transcriptase polymerase chain reaction analysis for KiSS-1 and orphan G-protein- coupled receptor (hOT7T175) gene expression in hepatocellular carcinoma. J Cancer Res Clin Oncol 2003, 129(9):531-535. 13. Makretsov N, Gilks CB, Coldman AJ, Hayes M, Huntsman D: Tissue microarray analysis of neuroendocrine differentiation and its prognostic significance in breast cancer. Hum Pathol 2003, 34(10):1001-1008. 14. Makretsov NA, Huntsman DG, Nielsen TO, Yorida E, Peacock M, Cheang MC, Dunn SE, Hayes M, van de Rijn M, Bajdik C et al: Hierarchical clustering analysis of tissue microarray immunostaining data identifies prognostically significant groups of breast carcinoma. Clin Cancer Res 2004, 10(18 Pt 1):6143-6151. 15. Parker RL, Huntsman DG, Lesack DW, Cupples JB, Grant DR, Akbari M, Gilks CB: Assessment of interlaboratory variation in the immunohistochemical determination of estrogen receptor status using a breast cancer tissue microarray. Am J Clin Pathol 2002, 117(5):723-728. 16. Wafa LA, Palmer J, Fazli L, Hurtado-Coll A, Bell RH, Nelson CC, Gleave ME, Cox ME, Rennie PS: Comprehensive expression analysis of L- dopa decarboxylase and established neuroendocrine markers in neoadjuvant hormone-treated versus varying Gleason grade prostate tumors. Hum Pathol 2007, 38(1):161-170. 17. Hofer MD, Browne TJ, He L, Skotheim RI, Lothe RA, Rubin MA: Identification of two molecular groups of seminomas by using expression and tissue microarrays. Clin Cancer Res 2005, 11(16):5722-5729. 18. Press JZ, De Luca A, Boyd N, Young S, Troussard A, Ridge Y, Kaurah P, Kalloger SE, Blood KA, Smith M et al: Ovarian carcinomas with genetic  77 and epigenetic BRCA1 loss have distinct molecular abnormalities. BMC Cancer 2008, 8:17. 19. Ng TL, Gown AM, Barry TS, Cheang MC, Chan AK, Turbin DA, Hsu FD, West RB, Nielsen TO: Nuclear beta-catenin in mesenchymal tumors. Mod Pathol 2005, 18(1):68-74. 20. Makretsov N, He M, Hayes M, Chia S, Horsman DE, Sorensen PH, Huntsman DG: A fluorescence in situ hybridization study of ETV6- NTRK3 fusion gene in secretory breast carcinoma. Genes Chromosomes Cancer 2004, 40(2):152-157. 21. Prentice LM, Shadeo A, Lestou VS, Miller MA, deLeeuw RJ, Makretsov N, Turbin D, Brown LA, Macpherson N, Yorida E et al: NRG1 gene rearrangements in clinical breast cancer: identification of an adjacent novel amplicon associated with poor prognosis. Oncogene 2005, 24(49):7281-7289. 22. Humphrey PA: Gleason grading and prognostic factors in carcinoma of the prostate. Mod Pathol 2004, 17(3):292-306. 23. Flechon A, Rivoire M, Droz JP: Management of advanced germ-cell tumors of the testis. Nat Clin Pract Urol 2008, 5(5):262-276. 24. Dhillo WS, Savage P, Murphy KG, Chaudhri OB, Patterson M, Nijher GM, Foggo VM, Dancey GS, Mitchell H, Seckl MJ et al: Plasma kisspeptin is raised in patients with gestational trophoblastic neoplasia and falls during treatment. Am J Physiol Endocrinol Metab 2006, 291(5):E878- 884. 25. Horikoshi Y, Matsumoto H, Takatsu Y, Ohtaki T, Kitada C, Usuki S, Fujino M: Dramatic elevation of plasma metastin concentrations in human pregnancy: metastin as a novel placenta-derived hormone in humans. J Clin Endocrinol Metab 2003, 88(2):914-919. 26. Weigelt B, Peterse JL, van 't Veer LJ: Breast cancer metastasis: markers and models. Nat Rev Cancer 2005, 5(8):591-602. 27. Gottsch ML, Cunningham MJ, Smith JT, Popa SM, Acohido BV, Crowley WF, Seminara SB, Clifton DK, Steiner RA: A role for kisspeptins in the regulation of gonadotropin secretion in the mouse. In: Endocrinology. vol. 145; 2004: 4073-4077. 28. Smith JT, Cunningham MJ, Rissman EF, Clifton DK, Steiner RA: Regulation of Kiss1 gene expression in the brain of the female mouse. In: Endocrinology. vol. 146; 2005: 3686-3692.  78 29. Adachi S, Yamada S, Takatsu Y, Matsui H, Kinoshita M, Takase K, Sugiura H, Ohtaki T, Matsumoto H, Uenoyama Y et al: Involvement of anteroventral periventricular metastin/kisspeptin neurons in estrogen positive feedback action on luteinizing hormone release in female rats. In: J Reprod Dev. vol. 53; 2007: 367-378. 30. Dhillo WS, Chaudhri OB, Patterson M, Thompson EL, Murphy KG, Badman MK, McGowan BM, Amber V, Patel S, Ghatei MA et al: Kisspeptin-54 stimulates the hypothalamic-pituitary gonadal axis in human males. J Clin Endocrinol Metab 2005, 90(12):6609-6615. 31. Moore CA, Milano SK, Benovic JL: Regulation of receptor trafficking by GRKs and arrestins. Annu Rev Physiol 2007, 69:451-482. 32. Yarden Y, Sliwkowski MX: Untangling the ErbB signalling network. Nat Rev Mol Cell Biol 2001, 2(2):127-137. 33. Brown LA, Hoog J, Chin SF, Tao Y, Zayed AA, Chin K, Teschendorff AE, Quackenbush JF, Marioni JC, Leung S et al: ESR1 gene amplification in breast cancer: a common phenomenon? Nat Genet 2008, 40(7):806- 807; author reply 810-802.    79 3 KISSPEPTIN AND GPR54 IMMUNOREACTIVITY IN A COHORT OF 518 PATIENTS DEFINES FAVOURABLE PROGNOSIS AND CLEAR CELL SUBTYPE IN OVARIAN CARCINOMA 2        Objective: To analyze GPR54 and kisspeptin by immunohistochemistry in ovarian cancer and determine any potential prognostic significance using linked clinical outcome data.          2   A version of this chapter has been published. Prentice LM, Klausen C, Kalloger S, Kobel M, McKinney S, Santos JL, Kenney C, Mehl E, Gilks CB, Leung P, Swenerton K, Huntsman DG, and Aparicio SAJ. Kisspeptin and GPR54 immunoreactivity in a cohort of 518 patients defines favourable prognosis and clear cell subtype in ovarian carcinoma. BMC Med. (2007);5:33.  80 3.1 Introduction The early diagnosis and management of ovarian cancer is a major area of unmet medical need. Central to the lack of progress in clinical management, has been the virtual absence of prognostic or predictive molecular markers for ovarian cancer. Key to addressing these questions is the availability of sufficiently large, clinically annotated tissue microarrays (TMA) that offer the prospect of defining the prognostic or predictive value of any given molecular marker. Therefore we have constructed a large ovarian cancer TMA (518 patients) with associated clinical demographic and outcome information and have used this to systematically address the value of possible biomarkers of disease prognosis. In the present study, we have tested the prognostic value of kisspeptin and GPR54 immunoreactivity in ovarian cancer. Kisspeptins (Kp-54, Kp-14, Kp-13, Kp-10) are the canonical, physiologically occurring and high affinity RF-amide peptide ligands that activate transmembrane signalling via a classical (7TM1) family G-protein coupled receptor, GPR54. Kisspeptins were first discovered through microcell mediated chromosome transfer experiments that defined the KiSS1 locus as a suppressor of melanoma tumour metastasis [1, 2]. Subsequently, kisspeptins were associated as endogenous ligands for the GPR54 receptor. Furthermore, a physiological role in the regulation of placental trophoblast invasion has been suggested [3] and in migratory cell lines, activation of GPR54 signalling abrogates migratory behaviour [1, 4-6]. In 2003 we uncovered in human and mouse genetic studies, the major physiological functions of kisspeptin-GPR54  81 signalling, as being gatekeepers for GnRH release in the hypothalamus [7, 8]. In the absence of functional kisspeptin [9] and GPR54 [8, 10-12] neither humans nor mice undergo puberty and are unable to generate pituitary release of gonadotropins that drive sex-steroid release. Several subsequent physiological studies have confirmed that kisspeptins act as neuroendocrine peptides that switch on or off the GnRH axis in humans and mammals [13-24] and are thus required as physiological regulators of sex-steroid release. The mechanistic relationship between GPR54 regulation of the hypothalamic-pituitary-gonadal axis, and possible effects on epithelial cell migration remains unclear, however several anecdotal studies on human tumours have suggested possible associations of loss/absence of expression, with poor prognosis [25-32].  Recently Zhang et al. [33] and Hata et al. [34] surveyed RNA expression of the KiSS1 and GPR54 loci in small cohorts (<100 cases) of ovarian cancer and observe a trend towards favorable prognosis where KiSS1/GPR54 RNA expression is elevated. None of these studies have been sufficiently powered to address cell-type and prognostic associations in major epithelial malignancies. We show in the present study of 518 ovarian cancer cases that kisspeptin and GPR54 immunoreactivity are very significantly associated with a clear cell carcinoma subtype and that both kisspeptins and GPR54 are independent markers for favourable prognosis as determined by multivariable analysis.   82 3.2 Material and Methods 3.2.1 Ovarian tumour samples and tissue microarray (TMA) construction Approval for the study was obtained from the ethics committee of the University of British Columbia. Ninety-three percent of women diagnosed with ovarian cancer in British Columbia are treated at the British Columbia Cancer agency (BCCA) and provincial treatment guidelines are followed. Outcomes are tracked via The Cheryl Brown Ovarian Cancer Outcomes Unit as an ovarian cancer database of the BCCA. A total of 3501 patients with invasive epithelial ovarian carcinoma were referred to the BCCA between 1984 and the year 2000. The focus of this study was 834 patients who had ovarian carcinoma with no macroscopic residual disease after surgery. For 202 cases the slides of the primary ovarian tumour were not available for review and these cases are excluded. A gynaecological pathologist (CBG) then performed a blinded full slide review of the remaining 632 cases. Tumour cell type and grade (Silverberg) were assessed; all clear cell carcinomas were considered to be grade 3, as per WHO recommendations. After review, 518 cases of invasive ovarian carcinoma were available in tissue blocks for tissue microarray construction. A representative area of each tumour was selected and a duplicate core tissue microarray (TMA) was constructed (Beecher Instruments, Silver Springs, MD); the cohort is described in Table 3.1.  Serial 4 m sections were cut for immunohistochemical (IHC) analysis.    83 Table 3.1 Clinicopathological characteristics of the cohort  Parameter N Histopathological Subtype Adenocarcinoma 4 Clear Cell 132 Endometrioid 125 Mucinous 31 Serous 212 Squamous Cell 1 Transitional 6 Undifferentiated 7 Grade 1 106 2 114 3 298 Stage 1 214 2 219 3 85  Age (years) Mean (SD) 58 (12.8) Median 57 Range (Min – Max) 25 - 89   84 3.2.2 Immunohistochemistry Kisspeptin Sections from formalin-fixed and paraffin-embedded tissues were deparaffinized with xylene and rehydrated with a graded series of alcohols.  Wet heat-induced antigen retrieval was performed in a steamer for 20 min with a modified citrate buffer (pH 6.1, Dako, Mississauga, ON). Following antigen retrieval, sections were treated with 3% H2O2 in PBS for 30 min to quench endogenous peroxidase activity. All of the aforementioned steps were followed by three washes with PBS for 5 min each. Slides were subsequently blocked for 30 min with serum-free protein block (Dako) and incubated overnight at 4°C with a polyclonal goat anti- KiSS-1 antibody (C-20, Santa Cruz Biotechnology, Santa Cruz, CA) diluted 1:400 in serum-free protein block.  Kisspeptin immunoreactivity (IR) was detected with the CSA II biotin-free tyramide signal amplification system and 3,3’- diaminobenzidine chromogen solution (Dako). Specifically, rabbit anti-goat-HRP was applied for 15 min followed by fluorescyl-tyramide amplification reagent for 15 min and anti-fluorescein-HRP for 15 min. All of the steps subsequent to the incubation with primary antibody were followed by three washes with TBST for 5 min each. Slides were counterstained with Harris hematoxylin (Sigma-Aldrich, Oakville, ON) and mounted in a xylene-based mounting medium. Based on previously published data showing cell-type restriction of GPR54 and kisspeptins in different trophoblast layers of human placenta [3], less than 10-week old human placenta was used as a specificity control (courtesy of Vancouver Coastal Health archives), in conjunction with two blocking peptides (21 residues and 54  85 residues – Figure 3.2). Omission of the primary antibody was used as a negative control.  GPR54 TMA 4 m sections were processed using a Ventana Discovery XT automated system (Ventana Medical Systems, Tucson, AZ) as per manufacturer’s protocol with proprietary reagents. After slides were baked at 60°C for 1 h, they were deparaffinized on the automated system with EZ Prep solution (Ventana). Heat- induced antigen retrieval method was used in Cell Conditioning solution (CC1- Tris based EDTA buffer, pH 8.0, Ventana).  The polyclonal rabbit GPR54 antibody was obtained from MBL International Corporation (Woburn, MA) specific for the N-terminal extracellular domain (catalogue number LS-A1929) and used with heat at a 1:25 concentration in Ventana antibody diluent. The Ventana Universal Secondary Antibody was used for 32 min at 37°C. The detection system used was the Ventana DABMap kit, and slides were then counterstained with Hematoxylin and treated with a proprietary bluing agent (Ventana).  All washes were conducted with the Ventana Reaction Buffer.  Dehydration steps and coverslip procedure were completed manually as per manufacturer’s recommendations. Specificity was determined by Western blot (Figure 3.1) and by using less than 10-week old human placenta as a positive control (Figure 3.2) and omission of primary antibody as a negative control.   86              Figure 3.1 Western blot demonstrating GPR54 specificity 30 μg of protein collected using the standard RIPA buffer method was run on 12% SDS-PAGE and transferred to a nitrocellulose membrane for detection with GPR54 MBL antibody for four cell lines. In three of four lanes a single band specific to GPR54 at 42kD is apparent. Loading control -actin is labeled.  87 3.2.3 Photomicrographs The TMA was digitally scanned with a BLISS (Bacus Laboratories Inc., Slide Scanner) automated system (Bacus Laboratories, Lombard, IL) as previously described [35]. These images are publicly available on our webslide server: http://www.gpecimage.ubc.ca/tma/web/viewer.php.  3.2.4 Statistical analysis Survival time dependant recursive partitioning was used to binarise the raw kisspeptin and GPR54 data.  Univariable survival analysis was performed by the generation of Kaplan-Meier curves [36] and differences between the groups were assessed using Log-rank Statistic [37].  Multivariable survival analysis was performed using the Cox Proportional Hazards Model [37, 38]; the adenocarcinoma, squamous cell, transitional, and undifferentiated ovarian subtypes were excluded from multivariable analysis due to insufficient sample size. Contingency tables and the Pearson Chi-square statistic were used to test the change in the distribution of kisspeptin and GPR54 expression across primary cell types [39].  All analyses were performed using JMP version 6.0.3 (SAS Institute, Cary NC, U.S.A.).   88 3.3 Results 3.3.1 Kisspeptin positivity is an independent marker for favourable prognosis Kisspeptin-IR was tested on human placenta less than 10 weeks old as a positive control (Figure 3.2).  Specifically, there was cell type specificity demonstrated by intense staining in syncytiotrophoblast cells as previously determined [3, 40], but not in other cell layers of the trophoblast. Pre-absorption with two different blocking peptides (metastin (Kp-54, 68-121aa) and kisspeptin 100-120aa), fully blocked kisspeptin-IR, whereas Kp-10 (112-121aa) showed little or no block (Figure 3.2). For the 518 case ovarian tissue microarray, kisspeptin-IR was scored as 0 for negative cases, +1 for mild staining, and +2 for intense staining (Figure 3.3). Of the 518 cases, 44 stained at +2, 98 had +1 staining intensity, 354 cases were negative for kisspeptin-IR, and 22 cases were uninterpretable.  The negative (0) and mildly reactive (+1) cases were grouped for statistical analysis and assigned the designation 0 and considered kisspeptin negative, while the +2 cases were considered kisspeptin positive and designated as 1.  Univariable disease specific survival analysis showed that kisspeptin-IR significantly associated with favourable prognosis (p=0.0023), as did overall survival (p=0.0006, Figure 3.4). Further, multivariable survival analysis including; stage, grade, histological subtype, age and GPR54-IR, indicated kisspeptin-IR as an independent marker for favourable prognosis in disease specific (p=0.0046, Table 3.2) and overall survival (p=0.0170, Table 3.3).  89    Figure 3.2 Immunohistochemistry controls Less than 10 week old human placenta used as a positive control. A) Kisspeptin- IR shows intense cell-type specific staining in the syncytiotrophoblasts (black arrow), while the cytotrophoblast layers remain unaffected (black arrowhead). B) GPR54-IR shows intense staining in the villous cytotrophoblasts (black arrow), the extravillous cytotrophoblasts (black arrowhead), and moderate staining on the syncytiotrophoblast membrane (grey arrow). C) Schematic of the 1-145 amino acid (aa) KiSS-1 pro-peptide. Metastin (Kp-54) is encoded within the 68- 121 aa sequence, while Kp-10 is encoded within this same region from 112-121 aa. The specific blocking peptide is encoded within the 100-120 aa sequence.  D)  90 Varying kisspeptin-IR was found among the different blocking peptides used. Blocking the primary antibody with full-length metastin (Kp-54) and blocking peptide resulted in complete loss of immunoreactivity, while Kp-10 was unable to block any detectable staining.  91     Figure 3.3 Immunoreactivity patterns Three types of kisspeptin-IR (A-C) and GPR54-IR (D-F) observed in the ovarian TMA. A, D) Representative samples of negative cases show complete lack of staining and are classified as 0. B) Moderate GPR54-IR shows a patchy staining pattern with light and dark brown regions of reactivity shown throughout the tumour core, and E) Mild kisspeptin-IR shows uniform light brown staining throughout the sample: both B and E are classified as +1 immunoreactivity. C, F) Examples of +2 intense immunoreactivity exhibit dark brown staining in all tumour cells. Scale bar represents 100 μm. Insets in each panel show a more detailed view of the staining pattern that is demonstrated in the larger image; inset scale bar represent 25 μm.   92 3.3.2 GPR54 positivity is an independent marker for favourable prognosis in overall survival The GPR54 antibody and protocol were tested on less than 10-week old human placenta and specifically stained both villous and extravillous cytotrophoblasts and the syncytiotrophoblasts as described by previous groups [3, 40] (Figure 3.2).  Although GPR54 is a 7-transmembrane protein, there was some reactivity in the cytoplasm of some tumour cells (this is not entirely surprising since GPR54 is a transmembrane protein and may be recycled through the cytoplasm) but only membranous staining was taken into consideration while assessing immunoreactivity.  Three immunoreactivity patterns were observed within the TMA for GPR54.  Specifically, negative or very weak reactivity in less than 5% of cells was designated as 0 (103 cases), while patchy or moderate staining in 5- 75% of cells was designated +1 (282 cases), and strong staining in greater than 75% of cells were considered +2 (104 cases, Figure 3.3).  The remaining 9 cases were uninterpretable.  As with kisspeptin, the 0 and +1 GPR54 cases were group together and considered as loss of receptor and designated 0, while the strong staining +2 cases were considered positive and designated as 1.  Univariable survival analysis determined GPR54 as a significant marker for favourable prognosis in disease specific (p=0.0092) and overall survival (p=0.0002, Figure 3.4).  Similar to kisspeptin-IR, GPR54 maintained significance in multivariable overall survival (p=0.0303, Table 3.3).  However, GPR54 was not found to be a significant independent marker in disease specific survival (p=0.1118, Table 3.2).    93 Figure 3.4 Disease specific and overall survival curves for Kp and GPR54 A) GPR54 expression in disease specific survival B) GPR54 expression in overall survival C) Kp expression in disease specific survival D) Kp expression in overall survival. E) Assessing both GPR54 and Kp expression in disease specific survival F) Assessing both GPR54 and Kp expression in overall survival. Cumulative survival is on the y-axis; time in years is on the x-axis. G+ represents GPR54 positive, G- represents GPR54 negative, K+ represents Kp positive cases, and K- represents Kp negative cases.  94     95  Table 3.2 Multivariable disease specific proportional hazards              *RR for age is not available because it is a continuous variable ** Represents statistical significance         Parameter Risk Ratio (95% CI) p-value Stage  <0.0001** 1 0.6404 (0.4901 to 0.8278) 2 0.7149 (0.5647 to 0.8994) 3 1.0000 Histological Grade  0.0720 1 0.6234 (0.3829 to 0.9808) 2 1.3899 (1.0193 to 1.8888) 3 1.0000 Subtype   0.2508 Clear cell   1.4519 (0.8853 to 2.3847) Endometrioid   0.6804 (0.4105 to 1.1032) Mucinous   1.0721 (0.5119 to 1.9699) Serous 1.0000 Age  0.0747  N/A* GPR54  0.1118 Positive 0.6475 (0.3738 to 1.1052) Negative 1.0000 Kisspeptin  0.0046** Positive 0.3508 (0.1426 to 0.7408) Negative 1.0000  96  Table 3.3 Multivariable overall proportional hazards               *RR for age is not available because it is a continuous variable ** Represents statistical significance         Parameter Risk Ratio (95% CI) p-value Stage  <0.0001** 1 0.7258 (0.5842 to 0.8961) 2 0.7149 (0.6457 to 0.9476) 3 1.0000 Histological Grade  0.5356 1 0.8268 (0.5818 to 1.1642) 2 1.1113 (0.8647 to 1.4148) 3 1.0000 Subtype   0.6763 Clear cell   1.2126 (0.7934 to 1.8513) Endometrioid   0.8120 (0.5508 to 1.1914) Mucinous   1.0192 (0.5652 to 1.6841) Serous 1.0000 Age  <0.0001**  N/A* GPR54  0.0303** Positive 0.5959 (0.3684 to 0.9523) Negative 1.0000 Kisspeptin  0.0170** Positive 0.4844 (0.2443 to 0.8841) Negative 1.0000  97 3.3.3 Kisspeptin positivity correlates with GPR54 positive cases Kisspeptin positive cases had a moderate correlation with GPR54 positivity as determined by Kendall’s tau-b [41] (=0.3837, p<0.0001).  There were 31 cases that were both kisspeptin and GPR54 positive, 90 cases that were kisspeptin negative and GPR54 positive, 12 cases with kisspeptin positivity and had loss of GPR54, 356 cases that had loss of both kisspeptin and GPR54, and the remaining 29 cases were uninterpretable.  When kisspeptin-IR and GPR54-IR cases are grouped together (G+ K+), patients have a more favourable outcome than those that have loss of either one or both (G- K+, G+ K-, G- K-).  There is a significant difference between survival for double positive patients (G+ K+) as compared to double negative patients (G- K-) in both disease specific (p=0.0067) and overall survival (p=0.0002, Figure 3.4).  3.3.4 Kisspeptin and GPR54 positive staining are significantly associated with clear cell carcinoma histo-pathological subtype The percentage of kisspeptin and GPR54 positive cases within each histo- pathological subtype is listed in Table 3.4. The proportionality of primary histo- pathological cell type in the entire cohort, kisspeptin positive cases, and GPR54 positive cases are represented in Table 3.5. Testing for an association between ovarian carcinoma subtype and kisspeptin status, there was a highly significant positive association with clear cell carcinoma, and a significant negative association with serous carcinoma subtype (2, p<0.0001).  GPR54 positive cases also had a significant positive association with clear cell carcinoma subtype and a negative association with the serous subtype (2, p<0.0001).  98  When disease specific survival and overall survival were analyzed within each ovarian carcinoma subtype, the Log-Rank test for kisspeptin-IR status failed to achieve significance (due to insufficient sample size), although for the clear cell cases statistical significance was approached (p=0.1042, p=0.0859, results not shown). Of note, none of the kisspeptin positive patients that were not clear cell subtype (16 cases) died from their disease. Similarly, when assessing GPR54 positivity within each ovarian cancer subtype, disease specific survival did not reach significance within the clear cell subtype (p=0.0656), although significance was achieved for overall survival (p=0.0102, Figure 3.5).  3.4 Conclusions Although clear cell carcinomas comprise fewer than 5% of ovarian malignancies, they are notoriously difficult to treat due to their tendency to resist platinum based chemotherapy [42]. To date, clinical stage has been the only prognostic marker for clear cell ovarian carcinoma. Here we show for the first time, that kisspeptin and GPR54 immunoreactivity are distinctly favourable prognostic markers, with kisspeptin being independent of pathologic subtype, stage, grade, or age in both overall and disease specific survival, while GPR54 is an independent marker in overall survival. Within clear cell carcinomas, GPR54 expressers have a favourable prognosis and to our knowledge this is the first molecular marker of prognosis specifically applicable to clear cell ovarian cancer. Although several studies have suggested possible relationships between GPR54 and kisspeptin expression and clinical outcome [25, 26, 29-33, 43], these studies have consisted  99 Table 3.4 Percentage of kisspeptin and GPR54 positive cases within the histological subtypes  Histological Subtype Kisspeptin Positive (%) GPR54 Positive (%) Clear Cell 21.88 66.41 Endometrioid   8.13 20.33 Mucinous 12.00 10.34 Serous   1.49   3.37   Table 3.5 Kisspeptin and GPR54 proportions within the cohort  Whole Cohort Kisspeptin Positive GPR54 Positive Histological Subtype Proportion Count Proportion Count Proportion Count Clear Cell 0.2640 132     *0.6364 28    **0.7131 87 Endometrioid 0.2500 125 0.2273 10 0.2049 25 Mucinous 0.0620 31 0.0682 3 0.0246 3 Serous 0.4240 212     *0.0682 3    **0.0574 7  *Chi square p-value<0.0001 **Chi square p-value<0.0001   100    Figure 3.5 GPR54 is a favourable prognostic marker in the clear cell subtype Kaplan-Meier survival curves for A) GPR54 expression in disease specific survival and B) GPR54 expression in overall survival. Cumulative survival on the y-axis; time in years on the x-axis.       101  of smaller cohorts and while some associations have been noted, some studies may not have  been sufficiently powered to address possible prognostic or cell type specific effects with rigor. To date, the present study is the largest systematic analysis of GPR54 and kisspeptin expression determined by immunoreactivity for an epithelial malignancy.  In part, this may be due to difficulties in obtaining sufficiently specific antisera and detection protocols, in that short peptides and GPCRs are notoriously difficult antibody targets. The antibodies and immunodetection protocols used in this study were verified by the use of either Western blotting and cell-type specific expression (GPR54), or cell- type specific expression and specific blocking peptides (kisspeptin). This is based on previous work showing differential expression of kisspeptin and GPR54 in human placental trophoblast cell types [3, 43]. The precise spectrum of immunoreactivity of the kisspeptin antiserum to kisspeptin fragments remains to be determined and, as with many antibodies, it remains possible that other proteins may be detected.  Very recently a survey of 76 ovarian cancer patients using Q-PCR detection of GPR54 and kisspeptin transcripts [34] demonstrated a negative correlation between KiSS-1 and GPR54 mRNA levels with residual disease, although they showed no correlation with histopathological subtype (possibly due to the relatively small number of clear cell ovarian cancers in that cohort), however the overall correlation observed in this study is in agreement with our observations. The mechanisms responsible for the association of kisspeptin and GPR54 expression with disease behaviour in ovarian cancer requires definitive studies,  102 however several possibilities arise.  It is possible that expression of kisspeptins and/or GPR54 result in higher endogenous GPR54 signalling in malignant cells. Although no studies have directly addressed the degree of GPR54 signalling in epithelial malignancies in relation to clinical outcomes, the present study shows that both kisspeptin and GPR54 expression are associated with a better prognosis. Furthermore, patients with double positive tumours (G+ K+) have the most favourable prognosis (Figure 3.3). These observations together with previous evidence of the effects of GPR54 signalling on cell migration, suggest some form of autocrine or paracrine loop could exist in clear cell carcinomas. GPR54 is exquisitely sensitive to kisspeptin ligand stimulation [3, 5] and receptor overexpression alone may be enough to increase basal signalling through GPR54. The interplay of mechanisms could be complicated by the major physiological role of GPR54, which is to regulate GnRH secretion at the hypothalamic level. Kisspeptins can cross from the peripheral circulation to act on the hypothalamus, as has been shown in numerous mammalian [21, 23, 44- 46] and one human study [20]. It is possible that kisspeptin overexpressing tumours may result in stimulation of the hypothalamic-pituitary axis, resulting in the release of gonadotropins and other derived peptides with a possible paracrine/endocrine effect on tumour growth.  Indeed Nash et al. [47], have shown that melanoma cells unable to signal on exposure to kisspeptins, can still be suppressed from metastasis by exogenous kisspeptin, suggesting that paracrine effects may operate in these cases. Finally, some evidence suggests  103 that kisspeptins and GPR54, which are expressed in ovarian epithelium and granulosa cells, may co-modulate the activity of gonadotropins in sex steroid release [40]. If such a mechanism were operational in clear cell ovarian cancers, it would imply that tumour behaviour is also linked to co-modulatory peptides.  Beyond the salient observation of prognostic significance in this study, the nature of the proteins involved suggests a number of possible areas for intervention. First, kisspeptins, the products of the KiSS-1 gene locus, are naturally occurring peptides that can be detected in human serum and other tissues [48-50]. It is possible that serum kisspeptide levels could be developed as a biomarker of disease activity in patients with clear cell carcinoma. Secondly, kisspeptins are naturally occurring peptide hormones that have activity in humans [20]. As such they are highly amenable to use as therapeutic agents, either alone or as modified peptides. We anticipate that the strong association of GPR54 and kisspeptin expression with outcome and clear cell type in ovarian carcinoma will stimulate fresh approaches to what is still a lethally intractable disease.  In conclusion, Kisspeptin and GPR54 are significantly associated with favourable prognosis in both disease specific and overall survival, as well as being significantly associated with the clear cell ovarian carcinoma subtype, thereby creating the first independent prognostic biomarkers specific for ovarian clear cell carcinomas.   104 3.5 References 1. Lee JH, Welch DR: Suppression of metastasis in human breast carcinoma MDA-MB-435 cells after transfection with the metastasis suppressor gene, KiSS-1. Cancer Res 1997, 57(12):2384-2387.  2. Lee JH, Miele ME, Hicks DJ, Phillips KK, Trent JM, Weissman BE, Welch DR: KiSS-1, a novel human malignant melanoma metastasis- suppressor gene. J Natl Cancer Inst 1996, 88(23):1731-1737.  3. Bilban M, Ghaffari-Tabrizi N, Hintermann E, Bauer S, Molzer S, Zoratti C, Malli R, Sharabi A, Hiden U, Graier W et al: Kisspeptin-10, a KiSS- 1/metastin-derived decapeptide, is a physiological invasion inhibitor of primary human trophoblasts. J Cell Sci 2004, 117(Pt 8):1319-1328 . 4. Ohtaki T, Shintani Y, Honda S, Matsumoto H, Hori A, Kanehashi K, Terao Y, Kumano S, Takatsu Y, Masuda Y et al: Metastasis suppressor gene KiSS-1 encodes peptide ligand of a G-protein-coupled receptor. Nature 2001, 411(6837):613-617.  5. Kotani M, Detheux M, Vandenbogaerde A, Communi D, Vanderwinden JM, Le Poul E, Brezillon S, Tyldesley R, Suarez-Huerta N, Vandeput F et al: The metastasis suppressor gene KiSS-1 encodes kisspeptins, the natural ligands of the orphan G protein-coupled receptor GPR54. J Biol Chem 2001, 276(37):34631-34636 . 6. Hori A, Honda S, Asada M, Ohtaki T, Oda K, Watanabe T, Shintani Y, Yamada T, Suenaga M, Kitada C et al: Metastin suppresses the motility and growth of CHO cells transfected with its receptor. Biochem Biophys Res Commun 2001, 286(5):958-963.  7. Aparicio SA: Kisspeptins and GPR54--the new biology of the mammalian GnRH axis. Cell Metab 2005, 1(5):293-296.  8. Seminara SB, Messager S, Chatzidaki EE, Thresher RR, Acierno JS, Jr., Shagoury JK, Bo-Abbas Y, Kuohung W, Schwinof KM, Hendrick AG et al: The GPR54 gene as a regulator of puberty. N Engl J Med 2003, 349(17):1614-1627.  9. d'Anglemont de Tassigny X, Fagg LA, Dixon JP, Day K, Leitch HG, Hendrick AG, Zahn D, Franceschini I, Caraty A, Carlton MB et al: Hypogonadotropic hypogonadism in mice lacking a functional Kiss1 gene. Proc Natl Acad Sci U S A 2007, 104(25):10714-10719.  10. Semple RK, Achermann JC, Ellery J, Farooqi IS, Karet FE, Stanhope RG, O'Rahilly S, Aparicio SA: Two novel missense mutations in g protein-  105 coupled receptor 54 in a patient with hypogonadotropic hypogonadism. J Clin Endocrinol Metab 2005, 90(3):1849-1855.  11. Funes S, Hedrick JA, Vassileva G, Markowitz L, Abbondanzo S, Golovko A, Yang S, Monsma FJ, Gustafson EL: The KiSS-1 receptor GPR54 is essential for the development of the murine reproductive system. Biochem Biophys Res Commun 2003, 312(4):1357-1363.  12. de Roux N, Genin E, Carel JC, Matsuda F, Chaussain JL, Milgrom E: Hypogonadotropic hypogonadism due to loss of function of the KiSS1-derived peptide receptor GPR54. Proc Natl Acad Sci U S A 2003, 100(19):10972-10976.  13. Patterson M, Murphy KG, Thompson EL, Patel S, Ghatei MA, Bloom SR: Administration of kisspeptin-54 into discrete regions of the hypothalamus potently increases plasma luteinising hormone and testosterone in male adult rats. J Neuroendocrinol 2006, 18(5):349-354.  14. Nazian SJ: Role of metastin in the release of gonadotropin-releasing hormone from the hypothalamus of the male rat. J Androl 2006, 27(3):444-449.  15. Castellano JM, Navarro VM, Fernandez-Fernandez R, Castano JP, Malagon MM, Aguilar E, Dieguez C, Magni P, Pinilla L, Tena-Sempere M: Ontogeny and mechanisms of action for the stimulatory effect of kisspeptin on gonadotropin-releasing hormone system of the rat. Mol Cell Endocrinol 2006, 257-258:75-83.  16. Arreguin-Arevalo JA, Lents CA, Farmerie TA, Nett TM, Clay CM: KiSS-1 peptide induces release of LH by a direct effect on the hypothalamus of ovariectomized ewes. Anim Reprod Sci 2006.  17. Seminara SB: Metastin and its G protein-coupled receptor, GPR54: critical pathway modulating GnRH secretion. Front Neuroendocrinol 2005, 26(3-4):131-138.  18. Navarro VM, Castellano JM, Fernandez-Fernandez R, Tovar S, Roa J, Mayen A, Barreiro ML, Casanueva FF, Aguilar E, Dieguez C et al: Effects of KiSS-1 peptide, the natural ligand of GPR54, on follicle-stimulating hormone secretion in the rat. Endocrinology 2005, 146(4):1689-1697.  19. Messager S, Chatzidaki EE, Ma D, Hendrick AG, Zahn D, Dixon J, Thresher RR, Malinge I, Lomet D, Carlton MB et al: Kisspeptin directly stimulates gonadotropin-releasing hormone release via G protein- coupled receptor 54. Proc Natl Acad Sci U S A 2005, 102(5):1761-1766.   106 20. Dhillo WS, Chaudhri OB, Patterson M, Thompson EL, Murphy KG, Badman MK, McGowan BM, Amber V, Patel S, Ghatei MA et al: Kisspeptin-54 stimulates the hypothalamic-pituitary gonadal axis in human males. J Clin Endocrinol Metab 2005, 90(12):6609-6615.  21. Thompson EL, Patterson M, Murphy KG, Smith KL, Dhillo WS, Todd JF, Ghatei MA, Bloom SR: Central and peripheral administration of kisspeptin-10 stimulates the hypothalamic-pituitary-gonadal axis. J Neuroendocrinol 2004, 16(10):850-858.  22. Navarro VM, Fernandez-Fernandez R, Castellano JM, Roa J, Mayen A, Barreiro ML, Gaytan F, Aguilar E, Pinilla L, Dieguez C et al: Advanced vaginal opening and precocious activation of the reproductive axis by KiSS-1 peptide, the endogenous ligand of GPR54. J Physiol 2004, 561(Pt 2):379-386.  23. Matsui H, Takatsu Y, Kumano S, Matsumoto H, Ohtaki T: Peripheral administration of metastin induces marked gonadotropin release and ovulation in the rat. Biochem Biophys Res Commun 2004, 320(2):383- 388.  24. Gottsch ML, Cunningham MJ, Smith JT, Popa SM, Acohido BV, Crowley WF, Seminara S, Clifton DK, Steiner RA: A role for kisspeptins in the regulation of gonadotropin secretion in the mouse. Endocrinology 2004, 145(9):4073-4077.  25. Shirasaki F, Takata M, Hatta N, Takehara K: Loss of expression of the metastasis suppressor gene KiSS1 during melanoma progression and its association with LOH of chromosome 6q16.3-q23. Cancer Res 2001, 61(20):7422-7425.  26. Sanchez-Carbayo M, Capodieci P, Cordon-Cardo C: Tumor suppressor role of KiSS-1 in bladder cancer: loss of KiSS-1 expression is associated with bladder cancer progression and clinical outcome. Am J Pathol 2003, 162(2):609-617.  27. Ringel MD, Hardy E, Bernet VJ, Burch HB, Schuppert F, Burman KD, Saji M: Metastin receptor is overexpressed in papillary thyroid cancer and activates MAP kinase in thyroid cancer cells. J Clin Endocrinol Metab 2002, 87(5):2399.  28. Nicolle G, Comperat E, Nicolaiew N, Cancel-Tassin G, Cussenot O: Metastin (KISS-1) and metastin-coupled receptor (GPR54) expression in transitional cell carcinoma of the bladder. Ann Oncol 2006.   107 29. Martin TA, Watkins G, Jiang WG: KiSS-1 expression in human breast cancer. Clin Exp Metastasis 2005, 22(6):503-511.  30. Ikeguchi M, Yamaguchi K, Kaibara N: Clinical significance of the loss of KiSS-1 and orphan G-protein-coupled receptor (hOT7T175) gene expression in esophageal squamous cell carcinoma. Clin Cancer Res 2004, 10(4):1379-1383.  31. Ikeguchi M, Hirooka Y, Kaibara N: Quantitative reverse transcriptase polymerase chain reaction analysis for KiSS-1 and orphan G-protein- coupled receptor (hOT7T175) gene expression in hepatocellular carcinoma. J Cancer Res Clin Oncol 2003, 129(9):531-535.  32. Dhar DK, Naora H, Kubota H, Maruyama R, Yoshimura H, Tonomoto Y, Tachibana M, Ono T, Otani H, Nagasue N: Downregulation of KiSS-1 expression is responsible for tumor invasion and worse prognosis in gastric carcinoma. Int J Cancer 2004, 111(6):868-872.  33. Zhang SL, Yu Y, Jiang T, Lin B, Gao H: [Expression and significance of KiSS-1 and its receptor GPR54 mRNA in epithelial ovarian cancer]. Zhonghua Fu Chan Ke Za Zhi 2005, 40(10):689-692.  34. Hata K, Dhar DK, Watanabe Y, Nakai H, Hoshiai H: Expression of metastin and a G-protein-coupled receptor (AXOR12) in epithelial ovarian cancer. Eur J Cancer 2007.  35. Ng TL, Gown AM, Barry TS, Cheang MC, Chan AK, Turbin DA, Hsu FD, West RB, Nielsen TO: Nuclear beta-catenin in mesenchymal tumors. Mod Pathol 2005, 18(1):68-74.  36. Kaplan E, Meier, P: Nonparametric estimation from incomplete observations. J Am Stat Assoc 1958, 53:457-481.  37. Cox D: Regression models and life tables. J R Stat Soc B 1972, 34:187-220.  38. Cox D: Partial likelihood. Biometrika 1975, 62:269-276.  39. Chernoff H, Lehmann, EL: The use of maximum likelihood estimates in chi square tests for goodness-of-fit. Ann Math Stat 1954, 25:576-586.  40. Castellano JM, Gaytan M, Roa J, Vigo E, Navarro VM, Bellido C, Dieguez C, Aguilar E, Sanchez-Criado JE, Pellicer A et al: Expression of KiSS-1 in rat ovary: putative local regulator of ovulation? Endocrinology 2006, 147(10):4852-4862.   108 41. Kendall M: Rank Correlation Methods. 4th edition. London: Griffin; 1970.  42. Pectasides D, Pectasides E, Psyrri A, Economopoulos T: Treatment issues in clear cell carcinoma of the ovary: a different entity? Oncologist 2006, 11(10):1089-1094.  43. Masui T, Doi R, Mori T, Toyoda E, Koizumi M, Kami K, Ito D, Peiper SC, Broach JR, Oishi S et al: Metastin and its variant forms suppress migration of pancreatic cancer cells. Biochem Biophys Res Commun 2004, 315(1):85-92.  44. Ramaswamy S, Seminara SB, Pohl CR, Dipietro MJ, Crowley WF, Jr., Plant TM: Effect of continuous iv administration of human metastin 45-54 on the neuroendocrine activity of the hypothalamic-pituitary- testicular axis in the adult male rhesus monkey (Macaca mulatta). Endocrinology 2007.  45. Plant TM, Ramaswamy S, Dipietro MJ: Repetitive activation of hypothalamic G protein-coupled receptor 54 with intravenous pulses of kisspeptin in the juvenile monkey (Macaca mulatta) elicits a sustained train of gonadotropin-releasing hormone discharges. Endocrinology 2006, 147(2):1007-1013.  46. Navarro VM, Castellano JM, Fernandez-Fernandez R, Tovar S, Roa J, Mayen A, Nogueiras R, Vazquez MJ, Barreiro ML, Magni P et al: Characterization of the potent luteinizing hormone-releasing activity of KiSS-1 peptide, the natural ligand of GPR54. Endocrinology 2005, 146(1):156-163.  47. Nash KT, Phadke PA, Navenot JM, Hurst DR, Accavitti-Loper MA, Sztul E, Vaidya KS, Frost AR, Kappes JC, Peiper SC et al: Requirement of KISS1 secretion for multiple organ metastasis suppression and maintenance of tumor dormancy. J Natl Cancer Inst 2007, 99(4):309- 321.  48. Panidis D, Rousso D, Koliakos G, Kourtis A, Katsikis I, Farmakiotis D, Votsi E, Diamanti-Kandarakis E: Plasma metastin levels are negatively correlated with insulin resistance and free androgens in women with polycystic ovary syndrome. Fertil Steril 2006, 85(6):1778-1783.  49. Horikoshi Y, Matsumoto H, Takatsu Y, Ohtaki T, Kitada C, Usuki S, Fujino M: Dramatic elevation of plasma metastin concentrations in human pregnancy: metastin as a novel placenta-derived hormone in humans. J Clin Endocrinol Metab 2003, 88(2):914-919.   109 50. Dhillo WS, Savage P, Murphy KG, Chaudhri OB, Patterson M, Nijher GM, Foggo VM, Dancey GS, Mitchell H, Seckl MJ et al: Plasma kisspeptin is raised in patients with gestational trophoblastic neoplasia and falls during treatment. Am J Physiol Endocrinol Metab 2006, 291(5):E878- 884.    110 4 PLASMA KISSPEPTIN IS ELEVATED IN GYNAECOLOGICAL MALIGNANCY 3         Objective: To analyze GPR54 and Kp IHC in an independent cohort of clear cell ovarian cancers and to determine if Kp can be detected in the plasma of ovarian cancer patients.           3  A version of this chapter will be submitted for publication. Prentice LM, Murphy KG, Kalloger S, Köbel M, Mehl E, Miller D, Gilks CB, Bloom SR, Huntsman DG, and Aparicio SAJ. Plasma kisspeptin is elevated in gynaecological malignancy.  111 4.1 Introduction Ovarian cancer has the highest number of cancer deaths of all the gynaecological cancers in North America [1, 2]. Many women unfortunately are diagnosed in the advanced stages of disease due to the lack of detectable symptoms in early stage cancer [3, 4]. Late stage directly correlates with poor prognosis [5] and early stage cancer is often treatable with surgery alone [3]. Although there is an ongoing search for biomarkers to reveal early stage ovarian cancer, few promising candidates have yet been unveiled. The metastasis suppressor kisspeptin (Kp) and its receptor GPR54 have been shown to have anti-metastatic effects in in vivo systems and anti-invasive characteristics when overexpressed in in vitro models [6-14]. Similarly, when gene expression is examined in cancer tissues KiSS1 and GPR54 are associated with less malignant disease and a more favourable prognosis [10, 13, 15-20]. In normal physiology, Kp and GPR54 are thought to mediate controlled invasion in placental trophoblast during pregnancy [21]. Indeed, the secreted Kp peptide can be measured in plasma and is at 10,000 fold higher levels in pregnant women, reaching its highest levels by the third trimester of pregnancy [22, 23]. Normal physiological levels of Kp are barely measurable in men and non-pregnant women (ranges from 1.3±0.1-6.9±1.9 pmol/L; [22-26]). Most recently, Kp was measured in the plasma of patients with malignant disease. Plasma Kp was elevated to near pregnant levels in women with gestational trophoblastic neoplasia (GTN); a malignant cancer of the placenta. Plasma Kp returned to barely detectable levels after surgery and chemotherapy  112 treatment of these patients; as is seen after the delivery of the placenta in normal physiology [22]. In pancreatic cancer, Kp was elevated in all patients compared to normal healthy volunteers, yet there was no correlation between plasma Kp and any prognostic markers such as: stage, grade, lymph node or distant metastasis [27]. However, clinical outcome as determined by survival analysis was not assessed. These initial studies suggest Kp may be a potential serum biomarker for cancer.  We have previously published Kp and GPR54 expression as being independent favourable prognostic markers in ovarian cancer, and specifically correlated expression to the clear cell cancer subtype [28]. Using a 445 case gynaecological cancer tissue microarray with matched plasma samples, we set out to determine Kp and GPR54 expression in these tumours and whether plasma Kp could also be measured. Here, we report that we have confirmed Kp and GPR54 specificity for the clear cell subtype in an independent cohort of ovarian cancers and for the first time determined that plasma Kp is elevated in gynaecological disease and cancer.  4.2 Material and Methods 4.2.1 Clear cell ovarian cancer arrays Two tissue microarrays consisting solely of clear cell cancer subtype cases with linked clinical outcome data were used. One TMA was from the Memorial Sloan- Kettering Cancer Center, New York, NY, USA consisting of 39 cases initially diagnosed as clear cell carcinoma obtained from patients from 1980-2006 [29].  113 The second TMA was from the University of Northern Alberta, Edmonton, Canada, consisting of 42 cases dating back to 1989 [30]. Characteristics of these two cohorts are listed in Tables 4.1 and 4.2.  4.2.2 Gynaecological Cancer Tissue Bank and array The Gynaecological Cancer Tissue Bank was formed to collect tissue from ovarian or uterine tumours from patients in British Columbia, Canada, and approval for the study was obtained from the ethics committee of the University of British Columbia. Patients undergoing oophorectomy or hysterectomy were asked to participate for one of the following reasons: (1) due to reasons unrelated to cancer, (2) due to disease which may be cancer, or (3) because the patient was at risk of developing cancer. After diagnostic pathological examination, remaining tissue was incorporated into the tissue bank, some of which had matched blood samples. Two gynaecological pathologists (Drs. Blake Gilks and Martin Köbel) performed a blinded full slide review of the cases to determine tumour cell type and grade (Silverberg). After review, 445 cases of gyneacological cancers were available in tissue blocks for tissue microarray construction.  A representative area of each tumour was selected and a duplicate core tissue microarray (TMA) was constructed as previously described [31- 33](Beecher Instruments, Silver Springs, MD); the cohort is described in Table 4.3. Serial 4 m sections were cut for immunohistochemical (IHC) analysis.  114 Table 4.1 Characteristics of the New York clear cell array  Parameter N Histopathological Subtype Clear Cell 39 Grade 3* 39 Stage 1 18 2 8 3 9 4 2 Age (years) Mean (SD) 54.17 (11.47) Median 54 Range (Min – Max) 31.0-82.0 *All clear cell ovarian cancers are Grade 3 Table 4.2 Characteristics of the Edmonton clear cell array  Parameter N Histopathological Subtype Clear Cell 42 Grade 3* 42 Stage 1 30 2 5 3 5 4 0 Age (years) Mean (SD) 55 (12.2) Median 54 Range (Min – Max) 37.0-91.0 *All clear cell ovarian cancers are Grade 3      115  Table 4.3 Characteristics of the gynaecological tissue microarray  Parameter N  Parameter N Histopathological Subtype 445  Stage 374 Endometrial 21  1 78 Endometrial Clear Cell 1  2 51 Endometrial Endometrioid  17  3 199 Endometrial Mixed  2  4 46 Endometrial Serous  1  Grade 323 Ovarian 337  1 43 Adenocarcinoma NOS 13  2 66 Borderline Brenner Tumor 1  3 214 Brenner Tumour 3  Age (Years) 445 Clear Cell 33  Mean (SD) 58.6 (14.5) Dysgerminoma 2  95%CI (LCI – UCI) 57.2 - 59.9 Endometrioid 29  Follow Up (Years) 402 Endometrioid Borderline 2  Mean (SD) 2.1 (1.6) Fibroma 1  95%CI (LCI – UCI) 1.9 - 2.3 Germinoma 1 Granulosa Cell Tumor 11 Gynandroblastoma 1 High-Grade Serous 218 Low-Grade Serous 9 Malignant Mixed Mullerian 10 Mucinous 8 Mucinous Borderline 2 Serous Borderline 17 Sertoli-Leydig Cell 1 Thecoma 2 Undifferentiated 10 Yolk sac tumor 3 Other 47 Fallopian Tube 3 Follicular Lymphoma 1 Leiomyosarcoma 4 Mesenchymal Neoplasia  1 Metastasis 32 Pleomorphic Sarcoma 2 Squamous Cell 2 Trophoblast Tumour 1 Vulvar Squamous 1    116 4.2.3 Blood sample collection Blood samples were drawn from patients that had fasted for at least 8 hours prior to phlebotomy. Whole blood was drawn into a 6 ml BD Vacutainer tube containing 10.8 mg of K2EDTA (BD Biosciences, Mississauga, ON) on the day of surgery prior to anesthetic induction. The blood was transferred to a 15 ml centrifuge tube and spun at 1000x g for 10 min at 4°C.  The plasma was drawn off, snap frozen and stored at -140°C.  Under optimal circumstances, the entire procedure took less than 30 min.  4.2.4 Kp detection in plasma A well established radioimmunoassay (RIA) with 100% cross reactivity to Kp-54, Kp-14, and Kp-10 and with <0.01% reactivity to other human RFamide peptides was used [22, 24, 25, 34]. In brief, 100 μl of plasma Kp competitively bound to the Kp specific GQ2 antibody against 125I-labeled Kp-54 in 700 μl of 0.07 M phosphate buffer, pH 7.2, containing 0.3% bovine serum albumin for 3 days at 4°C. Charcoal absorption was used to separate free and bound 125I-labeled Kp- 54 for Kp concentration determination. Each sample was measured in duplicate and concentrations yielding less than 10 pmol/l were considered as zero in statistical analysis.  4.2.5 Automated GPR54 and kisspeptin immunohistochemistry All of the TMAs were stained using an automated system for both GPR54 and Kp IHC. TMA 4m sections were processed using a Ventana Discovery XT  117 automated system (Ventana Medical Systems, Tucson, AZ) as per manufacturer’s protocol with proprietary reagents. After slides were baked at 60°C for 1 h, they were deparaffinized on the automated system with EZ Prep solution (Ventana). Heat-induced antigen retrieval method was used in Cell Conditioning solution (CC1- Tris based EDTA buffer, pH 8.0, Ventana). The polyclonal rabbit GPR54 antibody was obtained from MBL International Corporation (Woburn, MA) specific for the N-terminal extracellular domain (catalogue number LS-A1929) and used with heat at a 1:25 concentration in Ventana antibody diluent. The polyclonal goat KiSS1 antibody from Santa Cruz (Santa Cuz, CA) was incubated for 2 h without heat at 1:10 dilution. For GPR54, the Ventana Universal Secondary Antibody was used for 32 min at 37°C, while a rabbit anti-goat (Jackson ImmunoResearch, West Grove, PA) at 1:500 dilution was used for KiSS1.  The detection system used for GPR54 was the Ventana DABMap kit, while for KiSS1 the Ultra detection system (Ventana) was used. All slides were then counterstained with Hematoxylin and treated with a proprietary bluing agent (Ventana) and washes were conducted with the Ventana Reaction Buffer.  Dehydration steps and coverslip procedure were completed manually as per manufacturer’s recommendations. For the ovarian array with associated plasma, GPR54 was re-optimized and used at a 1:100 dilution for 2 h without using the Ultra detection system (Ventana). Specificity was determined by Western blot for GPR54 and by using less than 10-week old human placenta as a positive control for both GPR54 and Kp.  118 Omission of primary antibody was used as a negative control for both antibodies as published previously [28].  4.2.6 Photomicrographs The TMA was digitally scanned with a BLISS (Bacus Laboratories Inc., Slide Scanner) automated system (Bacus Laboratories, Lombard, IL) as previously described [28, 35]. These images are publically available on our webslide server: http://www.gpecimage.ubc.ca/tma/web/viewer.php.  4.2.7 Statistical Analysis Univariable survival analysis was performed by the generation of Kaplan-Meier curves [36] and differences between the groups were assessed using Log-rank Statistic [37]. Contingency tables and the Pearson Chi-square statistic were used to test the change in the distribution of kisspeptin and GPR54 expression across diagnostic group and IHC score [38].  Comparisons between groups for plasma Kp analysis were performed using ANOVA (analysis of variation) followed by a Tukey's test to account for multiple comparisons. All analyses were performed using JMP version 6.0.3 (SAS Institute, Cary NC, U.S.A.).   119 4.3 Results 4.3.1 Kp as a favourable marker for prognosis is variable between tissue microarrays Based on our initial findings that Kp and GPR54 are specific to the clear cell subtype of ovarian cancer, we used two additional clear cell cancer specific arrays with linked clinical outcome data to validate Kp and GPR54 as markers for favourable prognosis within this subtype. However, the results differed quite dramatically between the two clear cell cohorts. For the New York array Kp cases with an IHC score of 0 or +1 were grouped as negative and cases that had the most intense staining at a score of +2 were considered positive. Based on this grouping, Kp was determined to be a significant marker for favourable prognosis in disease specific survival as only one Kp positive patient succumbed to their disease (p=0.0383, Figure 4.1 A). Conversely the Edmonton array, regardless of how Kp IHC scoring was grouped, had no significant association with survival (Figure 4.1 C). As the majority of cases were positive for GPR54 in both cohorts, the prognostic significance could not be delineated in either TMA (Figure 4.1 B, D).  4.3.2 Kp and GPR54 are associated with clear cell subtype in an independent tissue microarray Using a gynaecological cancer tissue microarray consisting primarily of ovarian cancers (377/445) varying in subtype, Kp and GPR54 expression correlated with the clear cell subtype (p0.0001, Tables 4.4 and 4.5), validating our previous report [28]. For this analysis GPR54 IHC scores 0 and +1 were grouped as  120    Figure 4.1 Disease specific survival curves for Kp and GPR54 in ovarian cancer clear cell subtype A) Kp positivity is associated with favourable prognosis in disease specific survival in the New York array (18 +ve cases, 14 -ve cases).  B) GPR54 expression in the New York array (24 +ve cases, 10 -ve cases). C) Kp expression in the Edmonton array (10 cases for 0 group, 20 cases for 1 group, 10 cases for 2 group). D) GPR54 expression in the Edmonton array (38 +ve cases, 3 -ve cases). Cumulative survival is on the y-axis and time in years is on the x-axis.   121  Table 4.4 Percentage of kisspeptin and GPR54 positive cases within the histological subtypes.  Histological Subtype Kisspeptin Positive (%) GPR54 Positive (%) Clear Cell 46.15 78.57 Endometrioid 10.71 3.33 Mucinous 11.92 37.5 HG Serous 12.5 11.96 LG Serous 0 25    Table 4.5 Kisspeptin and GPR54 proportions within the cohort  Whole Cohort Kp Positive GPR54 Positive Histological Subtype Proportion Count Proportion Count Proportion Count Clear Cell 0.0989 28 0.3077* 12 0.4151* 22 Endometrioid 0.1060 30 0.0769 3 0.0189 1 Mucinous 0.0283 8 0.0000 0 0.0566 3 HG Serous 0.7385 209 0.5897 23 0.4717 25 LG Serous 0.0283 8 0.0256 1 0.0377 2 * Chi square p-value<0.0001         122 negative to represent GPR54 loss, and +2 cases were considered as positive as in our initial study. Kp was also grouped as previously described with +2 considered as Kp overexpression and IHC scores of 0 and +1 were grouped as negative. However, as this cohort of patients is relatively new in terms of clinical outcome, no significant correlations were made with survival due to limited follow-up time (95% confidence interval for follow-up time is 1.9-2.3 years).  4.3.3 Kp plasma concentration is elevated in women with ovarian cancer, endometrial cancer and benign gynaecological disease From the gynaecological tissue bank, 225 plasma samples were available for Kp analysis. As Kp has a half-life of 27.6 ± 1.1 minutes [24], and it has been previously established that samples processed within an hour of venipuncture provide optimal results with this assay [34] only samples that had been processed within an hour were used (184/225). Patients were categorized based on the state of their disease at the time of blood draw. These categories were: benign, history of cancer, metastasis, malignant, low malignant potential (LMP), and no disease. The benign group consisted of patients that had benign disease at the time of the blood draw, while patients who had previously been treated for cancer but did not have cancer at the time of blood draw were classified as having had a history of cancer, and patients that were negative for any disease were classified as no disease. Patients presenting with a metastasis to the ovary from a primary cancer outside of the gynaecological malignancies were grouped as metastasis and the rest of the patients were grouped as either malignant or low malignant potential to describe their cancer. A summary of the patients within  123 each group is listed in Table 4.6. Based on this classification system, women presenting with a metastasis to the ovary had significantly higher levels of plasma Kp than did patients that had been previously treated for cancer (Figure 4.2 A). As we have previously reported significant association of Kp expression with the clear cell subtype of ovarian cancer, the malignant group of patients was further divided into the five main subtypes of ovarian cancer: endometrioid, clear cell, mucinous, high-grade serous, and low-grade serous [39]. Although there was no significant association of Kp concentration with any of the subtypes the clear cell cases had the highest mean concentration (Figure 4.2 B). More cases would be required to reach statistical significance between subtypes, as there are currently only 7 clear cell cases. Nor was there a significant association with plasma Kp when correlated with age at the time of surgery (p=0.3889). Of the 445 cases on the gynaecological cancer tissue microarray, 190 cases had matched plasma. Of these, 79 samples were measured for plasma Kp concentration, resulting in 71/79 cases that had matched Kp results from the IHC analysis, and 76/79 cases that had GPR54 IHC results. There was not a significant association between Kp plasma concentrations and Kp IHC expression (p=0.2847), nor was there any correlation between Kp plasma concentration and GPR54 IHC expression (p=0.1422, Figure 4.3).  124 Table 4.6 Characteristics of plasma samples by group  Classification N No Disease 10 BRCA Mutation Carrier 10 Benign 43 Brenner Tumour 2 Endometriosis 10 Fibroma 6 Hyperplasia 2 Leiomyoma 4 Mature Teratoma 5 Mucinous Cystadenoma 4 Serous Cystadenoma 8 Struma Ovarii 1 Thecoma 1 Low Malignant Potential 9 Mucinous Borderline 3 Serous Borderline 6 Malignant 125 Adenocarcinoma NOS 3 Clear Cell 7 Dysgerminoma 2 Endodermal Sinus 1 Endometrial   44 Endometrioid 13 High-Grade Serous 33 Immature Teratoma 1 Leiomyosarcoma 1 Low-Grade Serous 6 Malignant Mixed Mullerian 7 Mucinous 4 Squamous Cell 2 Undifferentiated 1 Metastasis (Primary) 15 Breast 4 Carcinoid 1 Krukenberg 5 Lymphoma 2 Pseudomyxoma Peritoneii 1 Renal Clear Cell 1 Lung Small Cell 1 History of Cancer 18 Breast 13 Endometrial 4 Colon 1    125  Figure 4.2 Plasma Kp correlation with malignant potential and subtype A) Kp concentration in patients that have had a history of cancer is significantly different than patients who have developed a metastasis to the ovary (p=0.0258). B) Plasma Kp by ovarian cancer subtype. Description of groups is in Table 4.6. Hx=history, LMP=low malignant potential.  126   Figure 4.3 Plasma Kp compared to IHC expression Boxplots showing relationship between IHC score on the x-axis and plasma Kp concentration on the y-axis for Kp IHC scores (left) and GPR54 IHC scores (right).   127 4.4  Conclusions In our study we have determined in an independent cohort of patients that Kp and GPR54 are indeed markers for the ovarian cancer clear cell subtype. Furthermore, we have been able to measure plasma Kp in a variety of gyneacological diseases including malignancy. Although Kp has been measured in the plasma of gestational trophoblastic neoplasia [22] and pancreatic cancer patients [27], this is the first report of plasma Kp measurements in gynaecological disease and malignancy. We have also determined that Kp protein expression varies across tissue microarrays originating from different institutions, though these TMAs are all comprised of the clear cell cancer subtype. We previously reported that Kp overexpression in ovarian cancer is an independent marker for favourable prognosis and correlated to the clear cell subtype [28]. This was established by classifying the highest Kp expressors (IHC score of +2) as positive. Using this same classification system, we have determined that the highest Kp expressors are a marker for favourable prognosis in disease specific survival for a clear cell specific TMA. Curiously, in a second cohort of clear cell patients, regardless of Kp IHC grouping, there was no significance associated with survival. This may be due to the differences in tissue preservation that may affect the stability of Kp in paraffin. However, as the preservation methods utilized by these institutions are unknown, we cannot explore this theory further.  We have also determined that in the gynaecological array, the Kp IHC results were not consistent with the plasma Kp levels. This may suggest that the  128 tissue is creating more Kp than is being maintained in the cells. This may be possible if Kp is being rapidly secreted into the bloodstream directly after translation. Indeed this hypothesis was first proposed by Bilban et al. [21] who were able to detect the 145aa KiSS1 pro-peptide in cell lysates, but unable to detect the mature Kp54 peptide. Assuming this hypothesis, the cells should have Kp mRNA levels that match the plasma Kp concentrations. However, Torricelli et al. [40] compared KiSS1 mRNA expression in term placenta, pre-term placenta and placenta from term non-labour deliveries (caesarean section) and determined that there was significantly more KiSS1 mRNA in placentas that had been delivered vaginally. The highest KiSS1 mRNA levels were seen in the pre- term placentas, though all three types of deliveries had similar plasma Kp detected in the maternal blood. Unfortunately the antibody used in the EIA assay to measure the maternal plasma has since been shown to cross-react with neuropeptide FF [41] that is also secreted into the bloodstream [42]. Curiously, it is the metastasis group of patients that have a significantly higher level of plasma Kp as compared to those that had been previously treated for cancer. It is important to distinguish that the metastasis group does not consist of patients whose gynaecological cancer had metastasized, but of patients who had a primary non-gynaecological cancer that had metastasized to the ovary. It has been established that the ovary expresses Kp in rodents, and overexpresses Kp in cancer [28, 43]. Therefore this result could arise from the ovary or surrounding tissues expressing Kp as a means to control the invasiveness of the metastasis.  129  Currently CA125 is the most promising candidate as a serum biomarker for ovarian cancer. However, CA125 is only elevated in half of stage I cancers [44] and elevated in benign disease and normal physiological conditions such as pregnancy and menstruation [3, 45]. These characteristics make it a poor marker for early stage disease and have prevented its use as a diagnostic marker. However it is currently used to monitor disease progression as CA125 levels increase upon a secondary recurrence and remain stable in the absence of malignancy [3]. Unlike CA125, Kp is not elevated during menstruation, as Kp is barely detectable through all phases of the human female menstrual cycle [25]. It has also been established that after treatment and eradication of the initial cancer, plasma Kp rapidly drops back to barely detectable levels [22], demonstrating that in gestational trophoblastic disease Kp is highly specific for the disease. Furthermore, Kp is barely detectable in normal human male and female physiology outside of pregnancy [22-26]. However, similar to CA125, Kp is elevated in benign disease and pregnancy.  The current trend is to assess a panel of serum biomarkers to be used in conjunction with CA125 to monitor ovarian cancer progression to achieve higher specificity and sensitivity [46, 47]. We propose that Kp, although perhaps not a valid serum biomarker in itself, with further validation may be a valuable addition in a serum biomarker panel.  130 4.5 References 1. Canadian Cancer Society NCIoC: Canadian Cancer Statistics. In.: Statistics Canada; 2008. 2. Jemal A, Siegel R, Ward E, Murray T, Xu J, Thun MJ: Cancer statistics, 2007. CA Cancer J Clin 2007, 57(1):43-66. 3. Rosenthal AN, Menon U, Jacobs IJ: Screening for ovarian cancer. Clin Obstet Gynecol 2006, 49(3):433-447. 4. Williams TI, Toups KL, Saggese DA, Kalli KR, Cliby WA, Muddiman DC: Epithelial ovarian cancer: disease etiology, treatment, detection, and investigational gene, metabolite, and protein biomarkers. J Proteome Res 2007, 6(8):2936-2962. 5. Cannistra SA: Cancer of the ovary. N Engl J Med 2004, 351(24):2519- 2529. 6. Jiang Y, Berk M, Singh LS, Tan H, Yin L, Powell CT, Xu Y: KiSS1 suppresses metastasis in human ovarian cancer via inhibition of protein kinase C alpha. Clin Exp Metastasis 2005, 22(5):369-376. 7. Kotani M, Detheux M, Vandenbogaerde A, Communi D, Vanderwinden JM, Le Poul E, Brézillon S, Tyldesley R, Suarez-Huerta N, Vandeput F et al: The metastasis suppressor gene KiSS-1 encodes kisspeptins, the natural ligands of the orphan G protein-coupled receptor GPR54. In: J Biol Chem. vol. 276; 2001: 34631-34636. 8. Lee JH, Miele ME, Hicks DJ, Phillips KK, Trent JM, Weissman BE, Welch DR: KiSS-1, a novel human malignant melanoma metastasis- suppressor gene. J Natl Cancer Inst 1996, 88(23):1731-1737. 9. Lee JH, Welch DR: Suppression of metastasis in human breast carcinoma MDA-MB-435 cells after transfection with the metastasis suppressor gene, KiSS-1. Cancer Res 1997, 57(12):2384-2387. 10. Masui T, Doi R, Mori T, Toyoda E, Koizumi M, Kami K, Ito D, Peiper SC, Broach JR, Oishi S et al: Metastin and its variant forms suppress migration of pancreatic cancer cells. Biochem Biophys Res Commun 2004, 315(1):85-92. 11. Muir AI, Chamberlain L, Elshourbagy NA, Michalovich D, Moore DJ, Calamari A, Szekeres PG, Sarau HM, Chambers JK, Murdock P et al: AXOR12, a novel human G protein-coupled receptor, activated by the peptide KiSS-1. J Biol Chem 2001, 276(31):28969-28975.  131 12. Ohtaki T, Shintani Y, Honda S, Matsumoto H, Hori A, Kanehashi K, Terao Y, Kumano S, Takatsu Y, Masuda Y et al: Metastasis suppressor gene KiSS-1 encodes peptide ligand of a G-protein-coupled receptor. Nature 2001, 411(6837):613-617. 13. Shoji S, Tang XY, Umemura S, Itoh J, Takekoshi S, Shima M, Usui Y, Nagata Y, Uchida T, Osamura RY et al: Metastin Inhibits Migration and Invasion of Renal Cell Carcinoma with Overexpression of Metastin Receptor. In: Eur Urol. 2008. 14. Yoshioka K, Ohno Y, Horiguchi Y, Ozu C, Namiki K, Tachibana M: Effects of a KiSS-1 peptide, a metastasis suppressor gene, on the invasive ability of renal cell carcinoma cells through a modulation of a matrix metalloproteinase 2 expression. In: Life Sci. 2008. 15. Dhar DK, Naora H, Kubota H, Maruyama R, Yoshimura H, Tonomoto Y, Tachibana M, Ono T, Otani H, Nagasue N: Downregulation of KiSS-1 expression is responsible for tumor invasion and worse prognosis in gastric carcinoma. Int J Cancer 2004, 111(6):868-872. 16. Ikeguchi M, Yamaguchi K, Kaibara N: Clinical significance of the loss of KiSS-1 and orphan G-protein-coupled receptor (hOT7T175) gene expression in esophageal squamous cell carcinoma. Clin Cancer Res 2004, 10(4):1379-1383. 17. Ringel MD, Hardy E, Bernet VJ, Burch HB, Schuppert F, Burman KD, Saji M: Metastin receptor is overexpressed in papillary thyroid cancer and activates MAP kinase in thyroid cancer cells. J Clin Endocrinol Metab 2002, 87(5):2399. 18. Sanchez-Carbayo M, Capodieci P, Cordon-Cardo C: Tumor suppressor role of KiSS-1 in bladder cancer: loss of KiSS-1 expression is associated with bladder cancer progression and clinical outcome. Am J Pathol 2003, 162(2):609-617. 19. Shirasaki F, Takata M, Hatta N, Takehara K: Loss of expression of the metastasis suppressor gene KiSS1 during melanoma progression and its association with LOH of chromosome 6q16.3-q23. Cancer Res 2001, 61(20):7422-7425. 20. Stark AM, Tongers K, Maass N, Mehdorn HM, Held-Feindt J: Reduced metastasis-suppressor gene mRNA-expression in breast cancer brain metastases. J Cancer Res Clin Oncol 2005, 131(3):191-198. 21. Bilban M, Ghaffari-Tabrizi N, Hintermann E, Bauer S, Molzer S, Zoratti C, Malli R, Sharabi A, Hiden U, Graier W et al: Kisspeptin-10, a KiSS- 1/metastin-derived decapeptide, is a physiological invasion inhibitor of primary human trophoblasts. J Cell Sci 2004, 117(Pt 8):1319-1328.  132 22. Dhillo WS, Savage P, Murphy KG, Chaudhri OB, Patterson M, Nijher GM, Foggo VM, Dancey GS, Mitchell H, Seckl MJ et al: Plasma kisspeptin is raised in patients with gestational trophoblastic neoplasia and falls during treatment. Am J Physiol Endocrinol Metab 2006, 291(5):E878- 884. 23. Horikoshi Y, Matsumoto H, Takatsu Y, Ohtaki T, Kitada C, Usuki S, Fujino M: Dramatic elevation of plasma metastin concentrations in human pregnancy: metastin as a novel placenta-derived hormone in humans. J Clin Endocrinol Metab 2003, 88(2):914-919. 24. Dhillo WS, Chaudhri OB, Patterson M, Thompson EL, Murphy KG, Badman MK, McGowan BM, Amber V, Patel S, Ghatei MA et al: Kisspeptin-54 stimulates the hypothalamic-pituitary gonadal axis in human males. J Clin Endocrinol Metab 2005, 90(12):6609-6615. 25. Dhillo WS, Chaudhri OB, Thompson EL, Murphy KG, Patterson M, Ramachandran R, Nijher GK, Amber V, Kokkinos A, Donaldson M et al: Kisspeptin-54 stimulates gonadotropin release most potently during the preovulatory phase of the menstrual cycle in women. In: J Clin Endocrinol Metab. vol. 92; 2007: 3958-3966. 26. Katagiri F, Tomita K, Oishi S, Takeyama M, Fujii N: Establishment and clinical application of enzyme immunoassays for determination of luteinizing hormone releasing hormone and metastin. In: J Pept Sci. vol. 13; 2007: 422-429. 27. Katagiri F, Nagai K, Kida A, Tomita K, Oishi S, Takeyama M, Doi R, Fujii N: Clinical significance of plasma metastin level in pancreatic cancer patients. Oncol Rep 2009, 21(3):815-819. 28. Prentice LM, Klausen C, Kalloger S, Köbel M, McKinney S, Santos JL, Kenney C, Mehl E, Gilks CB, Leung P et al: Kisspeptin and GPR54 immunoreactivity in a cohort of 518 patients defines favourable prognosis and clear cell subtype in ovarian carcinoma. In: BMC medicine. vol. 5; 2007: 33. 29. Leitao MM, Jr., Boyd J, Hummer A, Olvera N, Arroyo CD, Venkatraman E, Baergen RN, Dizon DS, Barakat RR, Soslow RA: Clinicopathologic analysis of early-stage sporadic ovarian carcinoma. Am J Surg Pathol 2004, 28(2):147-159. 30. Kobel M, Xu H, Bourne PA, Spaulding BO, Shih Ie M, Mao TL, Soslow RA, Ewanowich CA, Kalloger SE, Mehl E et al: IGF2BP3 (IMP3) expression is a marker of unfavorable prognosis in ovarian carcinoma of clear cell subtype. Mod Pathol 2009, 22(3):469-475.  133 31. Makretsov N, Gilks CB, Coldman AJ, Hayes M, Huntsman D: Tissue microarray analysis of neuroendocrine differentiation and its prognostic significance in breast cancer. Hum Pathol 2003, 34(10):1001-1008. 32. Makretsov NA, Huntsman DG, Nielsen TO, Yorida E, Peacock M, Cheang MC, Dunn SE, Hayes M, van de Rijn M, Bajdik C et al: Hierarchical clustering analysis of tissue microarray immunostaining data identifies prognostically significant groups of breast carcinoma. Clin Cancer Res 2004, 10(18 Pt 1):6143-6151. 33. Parker RL, Huntsman DG, Lesack DW, Cupples JB, Grant DR, Akbari M, Gilks CB: Assessment of interlaboratory variation in the immunohistochemical determination of estrogen receptor status using a breast cancer tissue microarray. Am J Clin Pathol 2002, 117(5):723-728. 34. Ramachandran R, Patterson M, Murphy KG, Dhillo WS, Patel S, Kazarian A, Ghatei MA, Bloom SR: Preanalytical factors affecting RIA measurement of plasma kisspeptin. Clin Chem 2008, 54(3):615-617. 35. Ng TL, Gown AM, Barry TS, Cheang MC, Chan AK, Turbin DA, Hsu FD, West RB, Nielsen TO: Nuclear beta-catenin in mesenchymal tumors. Mod Pathol 2005, 18(1):68-74. 36. Kaplan E MP: Nonparametric estimation from incomplete observations. J Am Stat Assoc 1958, 53:457-481. 37. Cox D: Regression models and life tables. J R Stat Soc B 1972, 34:187-220. 38. Chernoff H, Lehmann, EL: The use of maximum likelihood estimates in chi square tests for goodness-of-fit. Ann Math Stat 1954, 25:576-586. 39. Kobel M, Kalloger SE, Boyd N, McKinney S, Mehl E, Palmer C, Leung S, Bowen NJ, Ionescu DN, Rajput A et al: Ovarian carcinoma subtypes are different diseases: implications for biomarker studies. PLoS Med 2008, 5(12):e232. 40. Torricelli M, Galleri L, Voltolini C, Biliotti G, Florio P, De Bonis M, Petraglia F: Changes of placental Kiss-1 mRNA expression and maternal/cord kisspeptin levels at preterm delivery. Reprod Sci 2008, 15(8):779-784. 41. Goodman RL, Lehman MN, Smith JT, Coolen LM, de Oliveira CV, Jafarzadehshirazi MR, Pereira A, Iqbal J, Caraty A, Ciofi P et al: Kisspeptin neurons in the arcuate nucleus of the ewe express both  134 dynorphin A and neurokinin B. In: Endocrinology. vol. 148; 2007: 5752- 5760. 42. Sundblom DM, Panula P, Fyhrquist F: Neuropeptide FF-like immunoreactivity in human plasma. Peptides 1995, 16(2):347-350. 43. Castellano JM, Gaytan M, Roa J, Vigo E, Navarro VM, Bellido C, Dieguez C, Aguilar E, Sanchez-Criado JE, Pellicer A et al: Expression of KiSS-1 in rat ovary: putative local regulator of ovulation? Endocrinology 2006, 147(10):4852-4862. 44. Zurawski VR, Jr., Knapp RC, Einhorn N, Kenemans P, Mortel R, Ohmi K, Bast RC, Jr., Ritts RE, Jr., Malkasian G: An initial analysis of preoperative serum CA 125 levels in patients with early stage ovarian carcinoma. Gynecol Oncol 1988, 30(1):7-14. 45. Ghaemmaghami F, Karimi Zarchi M, Hamedi B: High levels of CA125 (over 1,000 IU/ml) in patients with gynecologic disease and no malignant conditions: three cases and literature review. Arch Gynecol Obstet 2007, 276(5):559-561. 46. Nolen B, Marrangoni A, Velikokhatnaya L, Prosser D, Winans M, Gorelik E, Lokshin A: A serum based analysis of ovarian epithelial tumorigenesis. Gynecol Oncol 2009, 112(1):47-54. 47. Oikonomopoulou K, Li L, Zheng Y, Simon I, Wolfert RL, Valik D, Nekulova M, Simickova M, Frgala T, Diamandis EP: Prediction of ovarian cancer prognosis and response to chemotherapy by a serum-based multiparametric biomarker panel. Br J Cancer 2008, 99(7):1103-1113.    135 5 ENDOGENOUS GPR54 IN CANCER CELL LINES IS INTRACELLULAR AND DOES NOT SIGNAL THROUGH THE CANONICAL Gq PATHWAY4        Objective: To assess Kp-GPR54 signalling in malignant cells that endogenously express GPR54 by creating a stably transfected GPR54 knockdown cell line model.         4  A version of this chapter will be submitted for publication. Prentice LM, Huntsman DG, and Aparicio SAJ. Endogenous GPR54 in cancer cell lines is intracellular and does not signal through the canonical Gq pathway.  136 5.1 Introduction KiSS1 was so named due to the metastatic Suppressor Sequence that it encodes as well as a tribute to the city in which it was discovered; Hershey, Pennsylvania [1]. The translated peptide, originally named metastin, was first discovered to have anti-metastatic properties in a human melanoma cell line [1]. Since this discovery, there have been several in vitro cell line models created overexpressing either KiSS1 or GPR54 as a means to dissect the intracellular signalling pathway [1-10]. Though these cell line models have re-created the anti- metastatic properties originally observed, few studies have been able to determine the precise mechanism by which GPR54 signalling induces anti- metastatic behaviour. Two of the original discovery papers attributed the anti-invasive effects of Kp to formation of focal adhesions and stress fibers; this has since been confirmed by a third group [5, 8, 11]. Kisspeptin expression and activation of GPR54 has also been linked to decreased matrix metalloproteinase activity, suggesting that a metastatically favourable environment created by matrix metalloproteinases is prevented [10, 12, 13]. Finally, Kp stimulated GPR54 has been suggested to inhibit chemotaxis, specifically through the prometastatic G protein-coupled receptor CXCR4, as CXCR4 was unable to respond to its ligand, SDF-1, after treatment with Kp-10 [8, 14]. The detection of intracellular calcium release upon Kp activation of GPR54 has been consistent amongst research groups, suggesting that Kp-GPR54 signalling is mediated through a Gq coupled pathway [5, 7, 8]. Similarly, many of  137 these same groups have seen mitogen-activated protein kinase (MAPK) activation through the increased phosphorylation of the extracellular signal- regulated kinase (pERK) [5, 9, 15-17]. Some studies have demonstrated that phosphorylation of Akt is a downstream effect of GPR54 signalling [9, 16, 18], however this is less well established than MAPK activity. Though there have been many studies overexpressing either KiSS1 [4, 6, 10] or GPR54 [2, 3, 5, 7-9]; no studies have yet been published reporting the impact of down-regulation of either ligand or receptor. Furthermore, the few studies based on cancer cell lines with endogenous GPR54 are renal [11, 13], pancreatic [15], and thyroid [16]. Our group is the first to assess endogenous GPR54 in ovarian and breast cancer cell lines, and to establish a successful receptor knockdown model to assess intracellular GPR54 signalling upon Kp stimulation.  5.2 Material and Methods 5.2.1 Cell lines The MCF-7 breast cancer cell line (ATCC; HTB-22) was grown in Dubellco’s modified Eagle’s medium (DMEM; Stemcell Technologies, Vancouver, BC) supplemented with 15% fetal bovine serum (FBS; Gibco Invitrogen, Carlsbad, CA) while the MDA-MB-231 breast cancer cell line (ATCC; HTB-26) was grown in DMEM supplemented with 10% FBS. The ovarian cell lines TOV-21G (ATCC; CRL-11730, clear cell subtype), TOV-112D (ATCC; CRL-11731, endometrioid subtype), and OV90 (ATCC; CRL-11732, serous subtype) were all grown in a 1:1  138 mixture of MCDB 105 with 1.5 g/l sodium bicarbonate (Sigma-Aldrich, St. Louis, MA) and Medium199 (Stemcell Technologies, Vancouver, BC) supplemented with 15% FBS (Gibco Invitrogen, Carlsbad, CA). The 184 h-Tert cell line was an immortalized human breast epithelial cell line that was a generous gift from Dr. Sandra Dunn (Departments of Pediatrics, Experimental Medicine and Medical Genetics, University of British Columbia, Canada) and were grown in MEBM medium (Clonetics, San Diego, CA) supplemented with SingleQuots (Clonetics, San Diego, CA), 400 μg/ml neomycin (Gibco Invitrogen, Carlsbad, CA), 1 μg/ml transferrin (VWR, Mississauga, ON), and 1.25 mg/ml isoproteranol (Sigma- Aldrich, Oakville, ON). All cell lines were incubated at 37°C with 5% CO2. Except for the 184 h-Tert cells, all cells were purchased from the American Type Culture Collection (ATCC; Manassas, VA).  5.2.2 RNA extraction RNA was extracted from cell lines using Trizol (Invitrogen, Carlsbad, CA) as per manufacturer’s recommendations. After a phosphate buffered saline (PBS) wash, Trizol was added to the tissue culture plate and cells were incubated at room temperature (RT) for 5 min. Chloroform (Sigma-Aldrich, Oakville, ON) was added to the Trizol treated sample at 20% of the volume and mixed vigorously for 15 sec before a second RT incubation of 2-3 m. Samples were then centrifuged at maximum speed for 30 min at 4°C. The aqueous layer was pipetted into a new eppendorf tube without disruption of the organic layer. RNA was precipitated from the aqueous layer with an equal volume of isopropanol and incubated at RT  139 for 10 min. The samples were centrifuged at maximum speed for 10 min at 4°C. The RNA pellets were washed with 75% ethanol and centrifuged at half maximum speed for 5 min at 4°C. The RNA pellets were air dried before being resuspended in an appropriate volume of DNAse/RNAse free water. RNA concentrations and purity were measured with a NanoDrop spectrophotometer (Thermo Fisher Scientific, Wilmington, DE).  5.2.3 Reverse transcription One microgram of RNA was DNAse treated prior to reverse transcription to eliminate any contaminating genomic DNA from the sample. Samples were incubated at RT with DNAse I (Invitrogen, Carlsbad, CA) for 15 min before the addition of 25 mM EDTA (Invitrogen, Carlsbad, CA) and heated to 65°C for 10 min to stop the reaction as per manufacturer’s recommendation. Samples were then immediately put on ice to cool. Random hexamers at 250 ng/μl (Invitrogen, Carlsbad, CA), 10 μM dNTPs (Invitrogen, Carlsbad, CA), 400 U MMLV-Reverse transcriptase enzyme (Invitrogen, Carlsbad, CA), 0.1 M DDT (Invitrogen, Carlsbad, CA), reverse transcriptase buffer (250 mM Tris-HCI pH 8.3, 375 mM KCl, 15 mM MgCl2), 8 U RNase OUT enzyme, and water were added to the DNAse treated samples. Samples were incubated at 23°C for 10 min then incubated at 37°C for 1 h. The reaction was stopped by a 10 min 65°C incubation step.   140 5.2.4 Quantitative real-time PCR (QPCR) The Applied Biosystems (ABI) 7900HT Fast Real-Time System was used to amplify and detect KiSS1 and GPR54 mRNA transcripts (Applied Biosystems, Foster City, CA). Gene specific probe and primer sets were ordered as 20x target assays from ABI specifically covering exon boundaries for human KiSS1 (Hs00158486_m1) and GPR54 (Hs00261399_m1). The 18s rRNA subunit (Hs99999901_s1) expression levels at 1/250 dilution were used as an endogenous loading control before comparative normalization to one of the samples included in the analysis. The ABI software calculated the gene expression values using relative quantification (RQ=2-Ct).  5.2.5 Protein extraction and detection Cells at 70-85% confluency were washed 2x with PBS before addition of radioimmunoprecipitation assay (RIPA) lysis buffer (150 mM NaCl, 50 mM Tris HCl pH 7.5, 1 mM EDTA, 0.1% SDS, 1% Sodium deoxycholate (NaDOC), 1% Triton-X 100) with protease inhibitors (Roche, Laval, QC) and incubated on ice for 20 min. Cells were then scraped from the tissue culture dish and centrifuged at maximum speed for 10 min at 4°C. The pelleted cell debris was then removed from the supernatant. Protein concentrations were assessed using either the Bradford assay (Bio-Rad, Hercules, CA) or the BCA Protein Assay Kit (Pierce, Rockford, IL) using the microplate procedure. Protein was detected by one of two methods. The first method consisted of running samples on a discontinuous SDS-polyacrylamide gel after heating with 10x sample buffer (60 mM Tris-Cl pH  141 6.8, 2% SDS, 10% glycerol, 0.025% bromophenol blue) at 95°C for 5 min, followed by wet transfer to a polyvinylidene fluoride (PVDF) membrane (Millipore, Billerica, MA) that was blocked with Tris buffered saline with 0.5% Tween (TBST) containing 5% fat-free milk powder or 5% bovine serum albumin (BSA; pERK detection) at RT for 1 h. The membrane was then incubated overnight in blocking buffer with the primary antibody at 4°C followed by incubation with a horseradish peroxidase (HRP) conjugated secondary antibody at RT for 1 h prior to enhanced chemiluminescent (ECL) detection (Amersham Bioscience, Buckinghamshire, UK) or SuperSignal detection (Pierce, Rockford, IL) onto x-ray film (Kodak, Burnaby, BC). Alternatively, the ovarian cell protein lysates, were run on a 4-12% gradient NuPage Bis-Tris precast gel (Invitrogen, Carlsbad, CA) after protein lysate was incubated with 4x NuPage LDS sample loading buffer and 10x Reducing Agent (Invitrogen, Carlsbad, CA) at 70°C for 10 min followed by semi- dry transfer onto nitrocellulose using the iBlot system (Invitrogen, Carlsbad, CA). Similarly, these membranes were blocked at RT for 1 h in Odyssey blocking buffer (LI-COR Biotechnology, Lincoln, NB) and incubated overnight with the primary antibody in blocking buffer at 4°C, followed by incubation with either goat anti-mouse or goat anti-rabbit secondary antibody conjugated to an infrared (IR)- dye before being visualized with the LI-COR Odyssey infrared detection system (LI-COR Biotechnology, Lincoln, NB). Protein levels were quantified using intensity values provided by the LI-COR software. A ratio was obtained by comparing the intensity of the protein of interest to the loading control before being normalized to a control sample on the same blot.  142 5.2.6 Antibodies and peptides For the kisspeptins, two peptides were alternately used depending on the results of the assay. The literature argues that GPR54 has higher affinity for the smallest kisspeptin, Kp10, however there are many articles that have only found metastin as the biologically active peptide for GPR54 as discussed in Chapter 1. Consequently, if initial studies using one peptide were negative, then the second peptide was attempted. There were no differences between the two peptides throughout this chapter. Peptides used to stimulate the mitogen-activated protein kinase (MAPK) pathway were either; metastin (Kp-54 amide; Phoenix Pharmaceuticals, Burlingame, CA), kisspeptin-10 (Kp-10 amide; Phoenix Pharmaceuticals, Burlingame, CA) or epidermal growth factor (EGF: Sigma- Aldrich, Oakville, ON) as a positive control. Reagents used to stimulate the GPCR Gq pathway were Kp-10 and adenosine triphosphate (ATP; Sigma- Aldrich, Oakville, ON) as a positive control. Cells were stimulated with either Kp- 10 or Forskolin as a positive control for the cAMP assay. The antibodies that were used including their characteristics and concentrations are listed in summary Table 5.1.  5.2.7 Calcium mobilization assay Intracellular calcium was measured by a single-wavelength indicator (Fluor-4) to assess any rapid release of calcium, and also measured by a dual-wavelength indicator (Fura-2 AM) to account for any uneven indicator distribution and/or concentrations. For Fluor-4 (Molecular Probes, Eugene, OR) experiments, 2 x104  143 cells were plated into an Opti-well polysterene 96-well plate (Nalge Nunc, Rochester, NY) after 1:100 poly-L-lysine treatment, and incubated overnight at 37°C with 5% CO2. The cells were loaded with indicator and measured at 518 nm after a 444 nm excitation as per manufacturer’s protocol. Relative fluorescent units (RFU) were determined using the equation: RFU = Ft–Fo/Fo Where Ft was the averaged fluorescence emitted after treatment and Fo was the averaged fluorescence emitted before treatment. The RFU value for each cell type was averaged across three experiments. For dual-wavelength experiments, cells were plated at 40,000 cells/well in a glass-bottomed black-walled 96-well sterile tissue culture plate (Nalge Nunc, Rochester, NY). After a physiological saline solution (PSS; 10 mM HEPES, 5.9 mM KCl, 1.4 mM MgCl2, 1.2 mM NaH2PO4, 5 mM NaHCO3, 140 mM NaCl, 11.5 mM glucose, 18 mM CaCl2) wash, the cells were loaded with 4 μM Fura-2 AM (Invitrogen, Carlsbad, CA) at RT for 30 min. Cells were then washed and incubated in PSS at RT for an additional 30 min in the dark. Dual kinetic measurements were obtained at 340 nm (excitation 340 nm, emission 518 nm) and 380 nm (excitation 390 nm; emission 518 nm) using a Fluoroskan Ascent FL (ThermoLabsystem, Helsinki, Finland) for ratiometric analysis of calcium release. Background was measured from empty wells containing only PSS and Fura-2 AM and was subtracted from the 340 nm and 380 nm measurements before obtaining the 340/380 ratios. To determine basal calcium levels, five dual-kinetic measurements were made before the addition of 1 μM Kp10 or 1 μM ATP by injection. Twenty to sixty measurements with five-second intervals were  144 conducted after the injection of the stimulus. To calculate concentrations of released calcium, 10 μM ionomycin with 40 μM EGTA was added to the cells after the 60 measurements and the cells were measured again 80 times at 20 sec intervals. These final, stabilized EGTA measurements were considered the calcium-free measurement while addition of 100 mM CaCl2 in 10 μM ionomycin measured 60 times with 20 sec intervals was considered the calcium-bound measurement. Finally, 2 mM MnCl2 in 10 μM ionomycin was added to the cells and measured 60 times at 20 sec intervals for the background measurements to be subtracted from all previous measurements. The internal calcium concentration was calculated as: [Ca++]i nM =135((R-Rmin)/(Rmax-R))(F380/B380). Where 135 is the disassociation constant for Fura-2 at RT, and R is the ratio of the treatment (Kp-10 or ATP) being assessed. Rmin is the averaged ratio of the final three calcium-free measurements, while Rmax is the averaged ratio of the final three calcium-bound measurements. F380 is the averaged calcium-free 380 nm measurement and B380 is the averaged calcium-bound 380 nm measurement. For basal calcium concentrations, ratios were obtained prior to addition of stimulus, while max calcium concentrations where obtained after the addition of Kp or ATP. Each basal and max calcium concentration is the average of three individual experiments.  5.2.8 Cyclic AMP assay 2 x104 cells per well were plated out into a 96-well plate and incubated overnight at 37°C with 5% CO2. Cells were stimulated with either Kp-10 (0 nM, 1 nM, 10  145 nM, 100 nM, 1 μM, 10 μM) or 10 μM Forskolin, as a positive control, in a solution containing 50 μM isobutylmethylxanthine (IBMX). After a 30 min incubation at 37°C with the stimulus, cells were lysed and intracellular cAMP levels were measured by Biotrak competitive binding enzymeimmunoassay (EIA) as per manufacturer’s protocol (Amersham, Piscataway, NJ). Control wells that were not carried forward with the cAMP assay were assessed for complete cell lysis by trypan blue staining.  5.2.9 Flow cytometry Cells at 70-85% confluency were washed with PBS before a 3 min incubation at 37°C with 0.05% trypsin-EDTA (Gibco Invitrogen, Carlsbad, CA) to detach the adherent cells from the tissue culture plate. Detached cells were placed into cold Hank’s Balanced Salt Solution (Gibco Invitrogen, Carlsbad, CA) with 2% FBS (HF) and centrifuged at 1200 rpm for 5 min. Cells were then resuspended in 10 ml of HF and filtered using a 40 μm filter (BD Biosciences, Mississauga, ON) before counting live cells. Each treatment and control used 7x105 cells. The permeabilized cells were incubated on ice in 4% paraformaldehyde (Electron Microscopy Sciences, Hatfield, PA) for 20 min before addition of Trition-X 100 (Sigma-Aldrich, Oakville, ON) to a final concentration of 0.1% and incubated for a further 10 min. All cells were incubated with 10 μg of human IgG anti-CD32 (ATCC; Manassas, VA) in HF as a blocking solution for 30 min on ice. Cells were then incubated with 1:50 GPR54 MBL#29 antibody (Ab) on ice for 30 min prior to incubation with the FITC conjugated anti-rabbit secondary Ab (Jackson  146 ImmunoResearch, West Grove, PA) at 1:200 for 15 min on ice. Finally, cells were incubated with 1:1000 propidium iodide (PI) for 5 min before assessment by a FACsort cell sorter (BD Bioscience, San Jose, CA). Negative controls were: no primary Ab, GFP Ab as an isotype control, and PI only. Cells were gated by shape using side scatter versus forward scatter then assessed for PI levels and FITC levels to determine dead versus live cells and GPR54 expressors versus non-expressors.  5.2.10 Immunofluorescence Cells were plated onto sterile coverslips in a 6-well plate in normal growth media and incubated at 37°C with 5% CO2 overnight. Cells were washed 2x with PBS before incubation with 4% paraformaldehyde (Electron Microscopy Sciences, Hatfield, PA) at RT for 20 min. Cells were then washed 3x with PBS before permeabilization with 0.1% Triton-X (Sigma-Aldrich, Oakville, ON) for 5 min at RT. A 5 min RT incubation with 0.1 M Glycine was used to decreased background fluorescence, prior to 1 h RT incubation with blocking buffer (8% BSA, 5% human serum, 5% donkey serum in PBS). Coverslips were incubated overnight at 4°C with 1:100 GPR54 MBL#29 in blocking buffer before 1 h RT incubation with 1:200 FITC-conjugated donkey anti-rabbit (Jackson ImmunoResearch, West Grove, PA) in PBS with 1% BSA. 1:5000 Draq5 (Alexis Biochemicals, San Diego, CA) or 1:1000 PI was used as a nuclear stain and omission of the primary antibody was used as a negative control. Images were acquired using a fluorescent confocal microscope (Nikon Instruments,  147 Mississauga, ON) and accompanying software. For -arrestin co-localization studies, 1:100 -arrestin was used with 1:100 GPR54 MBL#29. Secondary antibody contained 1:200 of both donkey anti-rabbit conjugated with Cy-5 and donkey anti-goat conjugated to FITC. For co-localization of GPR54 with actin, an Oregon Green 488-conjugated phalloidin molecule was used (Molecular Probes, Invitrogen, Carlsbad, CA).  5.2.11 GPR54 shRNA knockdown After attempts at transient siRNA knockdown of GPR54 failed, a more stable knockdown method was used. Short hairpin RNAs for human GPR54 in pLKO.1 vectors were used to create stably transfected cell lines (shRNA11605, shRNA11606, shRNA1108, shRNA1109; Open Biosystems Thermo Fisher Scientific, Huntsville, AL). A pLKO.1 empty vector (Origene, Rockville, MD) and a pLKO.1 vector containing a non-specific scramble sequence (Addgene, Cambridge, MA) were used as negative controls. Hairpin containing vectors were purified from ampicillin resistant bacteria by an endotoxin free Maxi Prep kit as per manufacturer’s recommendations (Qiagen, Mississauga, ON). Vectors were restriction digested and sequenced by Sanger method to verify for accuracy. Cells were transfected using Lipofectamine 2000 (L2K; Invitrogen, Carlsbad, CA) as per manufacturer’s recommendations. Due to the variety of the cell lines used in this experiment (MCF-7, TOV21G, TOV112D, OV90), transfection and puromycin selection conditions were optimized for each cell line prior to knockdown experiments. Based on these optimizations each cell line was  148 transfected using at least two different L2K to DNA concentrations and assessed for successful GPR54 knockdown by western blot assay. The summary of these experiments is listed in Table 5.2. For transfected cells that successfully formed puromycin resistant clones, these colonies were pipetted from the plate into a new 24-well plate.  5.2.12 KiSS1 siRNA knockdown Transient knockdown of KiSS1 was performed as per manufacturer’s recommendations using three separate siRNAs (siRNA08, siRNA09, siRNA10; Dharmacon, Chicago, IL) in the TOV21G cells. Transfection methods for this cell line were based on successful lamin knockdown using Dharmacon reagents. Cells at 70% confluency in a 6-well plate were treated with either 50 nM DharmaFECT reagent 2 alone as a transfecting reagent control, 50 nM with siControl #2 as a non-specific target control, 50 nM of either of the KiSS1 siRNAs (08, 09, 10), and the last remaining well was left untreated. This experiment was done twice; once in eightfold for four time points (24 h, 48 h, 72 h, 96 h) to determine GPR54 localization by immunofluorescence (IF) with matched RNA samples to determine KiSS1 knockdown, and once in six fold for three time points (12 h, 36 h, 60 h) to determine GPR54 localization by flow cytometry with matched RNA samples to verify KiSS1 knockdown.  149  Table 5.1 Summary of antibodies used  Antibody Target Host Clonality Supplier Use Dilution -arrestin-1 (K-16) near C-terminus Goat Polyclonal Santa Cruz IF 1/100 IF 1/100 WB 1/1000 GPR54 LS-A1929 N-terminus Rabbit Polyclonal MBL International FC 1/50 pERK p44/p42 MAPK Rabbit Polyclonal Cell Signalling WB 1/1000 tERK (44/42 MAPK) clone 1B3B9 Mouse Monoclonal Upstate Biotechnology WB 1/5000 -actin N-terminus Mouse Monoclonal Sigma-Aldrich WB 1/5000 Vinculin entire protein Mouse Monoclonal Sigma-Aldrich WB 1/10000 IF=immunofluorescence, WB=western blot, FC=flow cytometry    Table 5.2 Summary of transfection conditions  Cell Line Puromycin L2K (ug/ml) Vector (ug/ml) Clones GPR54 KD MCF-7 400ng/ml 3.5 3.5 No No (p1)     4.5 3 No No (p1)     3.5 3 No ND TOV112D 600ng/ml 3.5 3.5 No No (p1)     4.5 3 No ND     6 3 Mixed No (p2)       C1 Yes (p3) TOV21G 2ug/ml 4.5 3.5 No No (p1)     6 3 Mixed No (p2)       C1 Unclear (p3)       C2 Yes (p3) OV90 1ug/ml 7 3.5 No No (p1)     8 4 No Yes (p2) L2K=lipofectamine 2000, KD=knockdown, p=passage number, ND=not determined  150 5.3 Results 5.3.1 GPR54 and Kp expression varies across a panel of cell lines To determine which cell lines would be best suited for in vitro functional studies of GPR54 signalling, it was imperative to determine endogenous expression levels of both the receptor and ligand across a panel of cell lines. In the ovarian cell lines, GPR54 protein was most highly expressed in the OV90 serous cell type, and least expressed in the TOV21G clear cell line (Figure 5.1 A). KiSS1 expression did not vary dramatically across the ovarian cell line subtypes and was most highly expressed in the MDA-MB-231 breast cancer cell line (Figure 5.1 B). Of the breast cell lines, GPR54 was most highly expressed in the MCF-7 cell line and the MDA-MB-231 cells had low expression (Figure 5.1 A). Of note, the immortalized normal epithelial mammary cell line 184 h-Tert did not express either GPR54 or KiSS1 and was used as an endogenous negative control for some of the studies.  RNA expression of GPR54 as determined by real-time PCR correlated well with the protein levels determined by western blot analysis (linear regression r2=0.94471). Only RNA expression of KiSS1 could be determined for the cell lines as a specific antibody that works for western blotting has not yet been made available.      151    Figure 5.1 Comparison of GPR54 and KiSS1 expression across cell lines A) Protein levels of GPR54 in the ovarian cell lines TOV21G, TOV112D, and OV90 as well as in breast cell lines, 184 h-Tert, MDA-MB-231, and MCF-7 by immunoblot analysis. B) Relative quantification of RNA levels of GPR54 and KiSS1 in the cell lines shown in (A), all normalized to the ovarian clear cell line TOV21G. Error bars represent standard error of the mean (SEM) across three replicates.  152  5.3.2 GPR54 does not signal through the Gq or the Gs signalling pathways upon Kp stimulation in breast cancer cell lines It has been previously reported that GPR54 signals through the Gq pathway that involves intracellular calcium release upon ligand binding. In order to assess receptor function in GPR54 expressing cell lines, we stimulated cells with either 1 μM Kp-10 or 1 μM Kp-54 and measured calcium release; as Kp-10 and Kp-54 have been previously shown to have equivalent effects on GPR54. Initial studies conducted in the MDA-MB-231, MCF-7, and 184 h-Tert breast cell lines showed no response to Kp-10 although the MCF-7 and 184 h-Tert cells responded strongly to 1 μM ATP (Figure 5.2 A). Confirming the presence of calcium stores capable of large release, the MCF-7 cells were responsive to 1 μM ATP and released 34.4 ± 3.3 nM of intracellular calcium. However, the addition of 1 μM Kp-10 did not significantly increase calcium levels above basal levels (Figure 5.2 B). To ensure that GPR54 was not signalling through an alternative G-protein coupled pathway, cyclic-AMP levels were measured as an indicator of Gs activation. Intracellular cAMP did not increase with 1 μM Kp-10 stimulation of the MDA-MB-231, MCF-7, or 184 h-Tert cells. At 10 μM of Kp-10, levels of cAMP increased in the MCF-7 cells from 18 fmol (at 0 nM Kp-10) to 51 fmol suggesting that super-physiological levels of Kp-10 could force signalling through the Gs pathway (Figure 5.2 C).   153 Figure 5.2 GPR54 does not signal through Gq or Gs in mammary cell lines A) 1 μM Kp-10 did not elicit a calcium flux from the MDA-MB-231, 184 h-Tert, or MCF-7 cell lines, although the 184 h-Tert and MCF-7 cells responded readily to 1 μM ATP as shown by increased relative fluorescence (RFU on the y-axis). B) The intracellular release of calcium ([Ca++]i on the y-axis) could be calculated above basal levels in the MCF-7 cells when stimulated with 1 μM ATP, but there was no detectable difference when stimulated with 1 μM Kp-10. C) Kp-10 stimulation of GPR54 did not signal through an alternate G pathway as determined by cAMP assessment (fmol cAMP on the y-axis) in the breast cell lines. Error bars represent ±SEM and are representative of three experiments in the calcium release assays (A and B), while they are indicative of duplicates in the cAMP study (C).     154   155 5.3.3 The MAP Kinase pathway is activated upon Kp stimulation of GPR54 expressing breast and ovarian cells Although the downstream effect of GPR54 Gq signalling is to activate the mitogen-activated protein kinase (MAPK) pathway, we asked whether MAPK activation could occur in the absence of Gq mediated calcium release. To measure MAPK activity, phosphorylated extracellular signal-regulated kinase (pERK) levels were determined by western blot after stimulation with Kp. The MCF-7 cells had a rapid increase of pERK (5 min) after stimulation with 1 μM Kp- 10, and this was dose dependent (Figure 5.3 A). Similar results were seen with the addition of 1 μM Kp-54 (results not shown). As 1 μM Kp gave reproducible results and was most often cited in the literature, this concentration was used for the rest of the MAPK activation experiments.  The MDA-MB-231 cells have increased endogenous levels of pERK due to a K-ras mutation that constitutively activates the MAPK pathway, thus GPR54 activity could not be determined upon the addition of 1 μM Kp-10 or 1 μM Kp-54, even under serum starved conditions (Figure 5.3 B). As expected, the GPR54 non-expressing 184 h-Tert cell line has no discernable levels of pERK after 1 μM Kp-10 or 1 μM Kp-54 stimulation (Figure 5.3 B).  For the ovarian cancer cell lines, which all express GPR54, there were increased levels of pERK upon 1 μM Kp-54 stimulation (Figure 5.3 C). Although the ovarian TOV21G clear cells express the lowest levels of GPR54, they had the highest upregulation of pERK upon Kp-54 stimulation; the highest fold   156   Figure 5.3 MAPK activation by Kp stimulation of GPR54 A) MAPK activation in MCF-7 cells at 5 min (left) and 15 min (right) after Kp-10 stimulation in serum starved conditions. The last lane shows pERK levels in normal medium without Kp-10 stimulation (no stv). B) MAPK activation in MDA- MB-231 cells (right) and 184 h-Tert cells (left) after stimulation with 1 μM Kp-10 in serum starved conditions. Last two lanes are Kp-10 untreated cells in either serum starved conditions (no tx stv) or in normal medium (no tx). C) MAPK  157 activation in ovarian cancer cell lines treated with 1 μM Kp-54 in serum starved conditions (top) compared to 10 ng/ml EGF used as a positive control. Vinculin is used as the loading control as tERK could not be determined due to a different blotting technique used in this assay, however tERK and vinculin levels were similar in these cell lines when assessed together in a separate experiment.   158 change was 2.33 5 min after treatment when compared to untreated cells. Comparatively, the TOV112D cells had a maximum fold increase of 1.63 at 5 min after treatment and the OV90 cells had a maximum fold increase of 1.82 at 10 min after treatment. As reproducible results were achieved with Kp-54 stimulation of the ovarian cancer cell lines, stimulation with Kp-10 was not performed. Cells were treated with 10 ng/ml EGF as a positive control.  Of note, p42 and p44 ERK can be distinguished in the ovarian cancer cells, and were not obvious in the breast cells. This is most likely due to the infrared detection system used for the ovarian cells whereas chemiluminescence was used to detect pERK signal in the breast cells. However, due to the infrared detection system, tERK could not be measured on the same blot as pERK and vinculin was used as a replacement. Vinculin and tERK were analyzed on the same immunoblot to ensure vinculin was an adequate substitute.  5.3.4 Knockdown of GPR54 is a temporary effect and does not affect pERK activation upon Kp10 stimulation After several unsuccessful attempts to knockdown GPR54 by transient short interfering RNAs (siRNA), a more permanent short hairpin (shRNA) method was used. Of the MCF-7, TOV21G, TOV112D, and OV90 cells transfected with the RNA hairpins, only the ovarian cell lines had successful knockdown of GPR54. All cell lines were transfected with three different hairpins (shRNA05, shRNA06, shRNA07) and at least two separate transfection parameters were used after lipofectamine:vector ratio was optimized. Of these four cell lines, only the three ovarian lines had detectable knockdown of GPR54 by western blot. Of these  159 three cell lines only the TOV21G and TOV112D successfully formed puromycin resistant clones. All the transfection parameters and results for each cell line are summarized in Table 5.2.  For the OV90 cells, at passage 2 (p2) after transfection, GPR54 was successfully knocked-down by 85% using shRNA06 as compared to untreated cells. However by p7, GPR54 protein levels only had 5% knockdown in the shRNA06 treated cells (Figure 5.4 A). For the TOV21G cells, shRNA05 and shRNA06 successfully knocked down GPR54 by p3 after transfection, and by p9 shRNA06 had maintained GPR54 knockdown by 88% as compared to untreated cells (Figure 5.4 B).  IF detection of GPR54 in the TOV21G cells confirmed that GPR54 protein was decreased in the shRNA05 and shRNA06 hairpin expressing cells at p9 (Figure 5.4 C). Having established prolonged knockdown of GPR54 in TOV21G cells, we investigated the consequences for pERK signalling at 1 μM Kp-10. Despite knockdown of GPR54, the TOV21G shRNA expressing cells and their controls all had upregulation of pERK upon 1 μM Kp-10 stimulation at p8 and again at p13 (Figure 5.4 D). Specifically the shRNA05 expressing cells had a maximum fold increase of 1.98 at 5 min after treatment while the shRNA06 expressing cells had a maximum fold increase of 1.94 at 5 min, compared to untreated cells. The TOV21G shRNA scramble control cells proliferated more quickly than the other transfected cells and increased cell confluency may account for raised basal levels of pERK seen in no tx, and 0 min control lanes.   160 Figure 5.4 Stable knockdown of GPR54 in TOV21G but not OV90 ovarian cells A) Immunoblot showing GPR54 expression in OV90 cells at passage 2 (p2; left) and p7 (right). Cells either received no treatment (no tx), scramble vector control (scram), empty vector control (empty), or a RNA hairpin specific for GPR54 (sh05 and sh06). B). Same experiment as described in (A) using the TOV21G ovarian cells at p3 (left) and p9 (right). -actin was used as a loading control in early passage (left) and vinculin was used as a loading control in later passage (right). C) FITC-labeled GPR54 (green) confirms knockdown in the shRNA05 and shRNA06 treated TOV21G cells as compared to the scramble and empty vector controls at passage 9. D) MAPK activation as determined by pERK in stably transfected TOV21G cells at p13. Cells are treated with 1 μM Kp-10 except for the no tx lanes; each lane represents time after Kp stimulation. p=passage, sh=short hairpin RNA  161   162 5.3.5 GPR54 is almost entirely cytoplasmic in MCF-7, TOV21G, TOV112D, and OV90 cells Although knockdown of GPR54 protein was observed in the TOV21G cells, we noticed that the GPR54 immunoreactivity was mostly cytoplasmic. Since GPR54 signalling through exogenous peptide normally requires a membranous receptor, we investigated the localization of GPR54 in the cancer cell lines. We used flow cytometry to determine if GPR54 was on the surface of the four cell lines, MCF-7, TOV21G, TOV112D, and OV90. We compared GPR54 expression of live intact cells to permeabilized cells, as live cells would only provide a signal for extracellular (membranous) GPR54 and permeabilized cells would reveal intracellular (cytoplasmic) GPR54.  The intact cells had low GPR54-FITC labeled signal indicative of minimal cell surface protein. Approximately one percent of the OV90 and theTOV21G cells had surface GPR54, while only 0.5% of the TOV112D and MCF-7 cells had plasma membrane GPR54 when compared to cells that had the GPR54 antibody omitted as a negative control (Figure 5.5). Of note, we were unable to locate a cell line with confirmed plasma membrane GPR54 to use as a positive control.  Comparatively, all cells had an approximately 10-fold increase in GPR54 signal after permeablization when compared to negative controls lacking the primary antibody; indicating a strong cytoplasmic signal for GPR54. Cytoplasmic GPR54 expression in ovarian cells compared to membranous expression is depicted in flow cytometry histograms in Figure 5.6 B.   163   Figure 5.5 Cell line expression of GPR54 on the plasma membrane as determined by flow cytometry Vertical axis measures propidium iodide (PI) signal indicative of cell death, while the horizontal axis measures FITC indicative of GPR54 signal. Percent of live cells expressing GPR54 are displayed in the lower right quadrant of each graph.  164 5.3.6 Intracellular GPR54 co-localizes with -arrestin but does not return to the membrane after knockdown of endogenous Kp in TOV21G cells G-protein coupled receptors may become internalized through phosphorylation of the intracellular C-terminus and subsequent -arrestin binding to the phosphorylated site. -arrestin can then signal for endocytosis of the receptor; either sequestering it to the cytoplasm or sending the receptor for protein degradation (as reviewed in [19, 20]). To determine whether GPR54 was co- localized with -arrestin, we performed dual IF in TOV21G cells (Figure 5.6 C). GPR54 was confirmed to be intracellular in TOV21G cells by IF when labeled actin revealed that the cell membrane did not co-localize with GPR54. However, cytoplasmic GPR54 could be detected (Figure 5.6 A). G-protein coupled receptors may become desensitized through continuous ligand binding which can lead to receptor internalization [20]. Since all four of the cell lines studied have KiSS1 expression (Figure 5.1), we hypothesized that endogenous Kp may be desensitizing GPR54. Therefore, we decreased KiSS1 transcripts by siRNA in the TOV21G cells to assess if GPR54 was being desensitized by autocrine Kp expression. However, the knockdown of KiSS1 in TOV21G cells did not shift GPR54 from the cytoplasm back to the membrane as determined by flow cytometry and IF (Figure 5.7).   165    Figure 5.6 GPR54 is intracellular and co-localizes with  -arrestin in TOV21G cells A) -actin outlines the membrane of the TOV21G cell (green); GPR54 is shown in red. B) Histogram depicting flow cytometry results for GPR54 signal (solid line) in ovarian cancer cells as compared to negative control without GPR54 primary  166 antibody (broken line). FITC signal representative of GPR54 expression is on the x-axis. C) Dual IF labeling of -arrestin in green and GPR54 in red in TOV21G cells. Co-localization is determined by a yellow signal resulting from the overlap of the two proteins depicted in the merge panel.   167 Figure 5.7 Localization of GPR54 is not altered by knockdown of endogenous Kp A) KiSS1 expression as determined by QPCR analysis in untreated cells (no tx), L2K only treated cells (mock), negative siRNA control treated cells (neg) and Kp siRNA treated cells (si06, si09, si10); error bars represent ±SEM across three technical replicates. Expression was measured at four timepoints from treatment (24 h, 48 h, 72 h, and 96 h) and is measured as relative quantification (RQ; y- axis). B) GPR54 protein is shown in green as determined by IF at 72 h after treatment with the negative siRNA control (neg; top) and siRNA 09 (bottom). C) Similar experiment as in (A) only with three timepoints (12 h, 36 h, and 60 h) and using only one siRNA. D) Determination of GPR54 localization by flow cytometry for cells treated in (C). Top three timepoints demonstrate GPR54 membranous signal as compared to cytoplasmic GPR54 (bottom). FITC signal that is specific to GPR54 is measured on the x-axis.   168    169 5.4 Conclusions The initial objective was to find breast and/or ovarian cancer cell lines expressing GPR54 to investigate the effect of abrogating GPR54 signalling. While the effects of kisspeptin signalling through endogenous GPR54 has been explored in trophoblast cells [12], renal cancer cells [11, 13], pancreatic cancer cells [15], and thyroid cancer cells [16] most have been engineered to overexpress GPR54 [2, 3, 5, 7-9].  Potent small molecule inhibitors of GPR54 have yet to be discovered, necessitating the use of RNA interference to knock down the expression of GPR54. After screening several cell lines, we found expression of GPR54 detectable by western blot, in two breast and three ovarian cancer cell lines.  Several studies have shown that GPR54 signals through the Gq pathway [5, 7, 8, 18, 21, 22]. Canonical GPCR signalling through G-proteins coupled to the Gq subunit results in intracellular calcium release and this was used to assay the responses of the cell lines to Kp-10 and Kp-54 (metastin) peptides. It takes as little as 6.4 nmol/kg Kp-54 to elicit significant release of FSH and LH in women in vivo [23]. Although most in vitro studies use 100 nM to1 μM of Kp when assessing GPR54 function in cell lines [3, 13, 21], concentrations as low as 10 nM have shown to elicit a response [24]. Engineered GPR54 overexpressing cell lines have exhibited an EC50 as low as 0.1 nM of Kp [5, 7, 8]. None of the breast cell lines investigated in our study manifest a calcium flux response, even when stimulated in the micro molar range, well above the physiological level at  170 which kisspeptins are thought to act, although all were capable of releasing calcium in response to ATP.  In the absence of Gq-protein mediated signalling, we next asked whether activation of the MAPK component ERK could be observed. GPCR mediated ERK activation can occur through Gs-proteins, G heterodimers, or through the non G-protein -arrestin pathway [19, 20, 25-27]. After Kp stimulation of the breast cell lines, we found dose dependent ERK phosphorylation in MCF-7 cells; the highest of the GPR54 expressing cells. The malignant MDA-MB-231 breast cell line showed high endogenous pERK even after serum starvation and careful washing to remove any potential mitogens, and thus could not be assessed further. The ovarian cancer cell line TOV21G exhibited measurable transient pERK1/2 increase at 1 μM Kp-54, despite having the lowest apparent level of expression of GPR54 by western blot. The OV90 cell line expressed the greatest amount of GPR54 amongst the ovarian cell lines, and showed an intermediate pERK1/2 response. Having shown that GPR54 was expressed in these lines and that a pERK1/2 response could be seen, albeit at super-physiological levels in the ovarian cell lines, we attempted to knockdown GPR54 using RNA mediated interference. We screened several independent shRNA hairpins in each of the MCF-7 and ovarian cell lines and were able to obtain knockdown of GPR54 protein in the ovarian cells. The shRNA06 hairpin was capable of mediating stable knockdown of GPR54 in the TOV21G cells, by 88% compared to untreated cells, and transiently stable knockdown in OV90. Though there was  171 evidence of GPR54 knockdown in the TOV112D cells, they were not suitable for IF and were not carried forward for further analysis. No measurable knockdown of GPR54 was observed in MCF-7 cells with the same shRNAs. We investigated whether the knockdown in the TOV21G cell lines resulted in abrogation of signalling. Despite measurable knockdown of protein, no effect on pERK1/2 response to Kp-10 was observed. Since we could not achieve sufficient stable knockdown of GPR54 to affect any change in pERK1/2 responses to Kp, and since Ca++ signalling was also not observed, we concluded that the original aim of altering endogenous GPR54 signalling to observe responses could not be assayed with the breast and ovarian cancer cells. The lack of knockdown effect on signalling could be due to a small number of remaining GPR54 receptors or due to Kp-10 acting through other receptors in the cells. The possibility of additional kisspeptin receptors has been suggested in the literature [28, 29], but to date without a candidate having been identified. During screening of the cell lines for GPR54 expression by IF, we noticed that the predominant localization of expression in TOV21G cells appeared to be cytoplasmic. We confirmed this in four cell lines (TOV21G, TOV112D, OV90, and MCF-7) by using flow cytometry to estimate the relative levels of GPR54 immunoreactivity. Fewer than 1% of live cells showed surface immunoreactivity for GPR54 whereas intracellular GPR54 immunoreactivity was approximately 10 fold above background. The cytoplasmic localization of GPR54 prompted us to ask whether GPR54 was trapped with -arrestin, a known alterative signalling and  172 sequestration compartment for GPCRs [19, 20]. Dual IF of GPR54 and -arrestin showed co-localization of the two proteins in the cytoplasm. Arrestins can sequester GPCRs in the cytoplasm and thereby downregulate activity of a signalling pathway [19, 20]. This can occur as a physiological response to chronic ligand stimulation of GPCRs, resulting in internalization. In fact, this process is exploited by GnRH agonists that operate on the GnRH receptor, through desensitization and downregulation of surface expression by chronic stimulation [30]. Finally, we decided to test the hypothesis that autocrine Kp released by the cell lines might be causing desensitization of the cells by -arrestin mediated trapping of internalized receptor. A siRNA construct capable of knocking down endogenous Kiss1 transcript by 90% as compared to controls resulted in no effect on co-localization of GPR54. In Chapter 2 of this dissertation it was observed that Kiss1 transcripts do not always match protein Kp as determined by IHC. We concede that Kiss1 knockdown may not reflect true Kp protein levels, yet as no antibody suitable for immunoblot detection of Kp exists, we are unable to verify downregulation of protein Kp.  Although signalling through the MAPK cascade can be observed in the breast and ovarian cells, it is not clear whether this is through an alternative GPR54 mediated -arrestin pathway or through a different receptor. The predominantly cytoplasmic expression of GPR54 suggests that the canonical membrane signalling arrangement does not occur in these cells. However, there are reports of endogenously internalized GPCRs that are still responsive to their  173 agonists, albeit the literature reports mutations in these GPCRs [31, 32]. There has yet to be a report of GPR54 mutations in cancer and we were unable to successfully sequence GPR54 in our cell lines. Thus, we acknowledge that an as yet unknown GPR54 mutation may exist and these results should be interpreted with caution.  This proposed mechanism of GPR54 activation bypassing the canonical Gq calcium release may be specific for endogenously expressing GPR54 cancer cell lines. Indeed, there has been the recent discovery of cell specific GPR54 signalling in neurons. Inhibition of ERK1 and ERK2 in the hippocampus prevented neuronal activity [33], while this same inhibitor did not affect neuronal activity in the hypothalamus after Kp treatment [34]. Calcium flux has been determined in endogenous GPR54 expressing primary trophoblast cells [12] and many in vitro overexpressing models [2, 3, 5, 8, 9], but this is the first reported case of an endogenously expressing cancer cell line that is unable to release calcium upon Kp stimulation. Other studies that have used endogenous GPR54 expression in cancer cell lines did not measure intracellular calcium release or determine GPR54 localization [11, 15, 16]. The major limitations of this study are specific to the functional experiments used in assessing GPR54 signallling. Firstly, a cell line expressing GPR54 on the plasma membrane would be a more appropriate positive control in the flow cytometry and the activation of MAPK studies. Similarly such a cell line would have been an appropriate control for the calcium flux analysis. If there was calcium flux using a membrane bound GPR54 cell line, we could infer that the  174 lack of calcium flux in the MCF-7 cell lines is specific to GPR54 localization. Unfortunately we did not have access to such a cell line. The second limitation of this study is the lack of a negative control specific to the Kp ligand. Although the Kp peptides used in these studies had the required amidation group at the N- terminus, by modifying the Kp peptide through removal of the amidation group, we could have determined that the Kp used in our studies was indeed active. This research demonstrates for the first time the endogenous GPR54 intracellular localization in four individual cancer cell lines. Arguably these results question the significance of relating GPR54 alone, to physiological effects or tumour cell behaviour. Clearly the membrane location and ability to signal should be taken into account in future studies.   175 5.5 References 1. Lee JH, Miele ME, Hicks DJ, Phillips KK, Trent JM, Weissman BE, Welch DR: KiSS-1, a novel human malignant melanoma metastasis- suppressor gene. J Natl Cancer Inst 1996, 88(23):1731-1737. 2. Becker JA, Mirjolet JF, Bernard J, Burgeon E, Simons MJ, Vassart G, Parmentier M, Libert F: Activation of GPR54 promotes cell cycle arrest and apoptosis of human tumor cells through a specific transcriptional program not shared by other Gq-coupled receptors. In: Biochem Biophys Res Commun. vol. 326; 2005: 677-686. 3. Hori A, Honda S, Asada M, Ohtaki T, Oda K, Watanabe T, Shintani Y, Yamada T, Suenaga M, Kitada C et al: Metastin suppresses the motility and growth of CHO cells transfected with its receptor. Biochem Biophys Res Commun 2001, 286(5):958-963. 4. Jiang Y, Berk M, Singh LS, Tan H, Yin L, Powell CT, Xu Y: KiSS1 suppresses metastasis in human ovarian cancer via inhibition of protein kinase C alpha. Clin Exp Metastasis 2005, 22(5):369-376. 5. Kotani M, Detheux M, Vandenbogaerde A, Communi D, Vanderwinden JM, Le Poul E, Brézillon S, Tyldesley R, Suarez-Huerta N, Vandeput F et al: The metastasis suppressor gene KiSS-1 encodes kisspeptins, the natural ligands of the orphan G protein-coupled receptor GPR54. In: J Biol Chem. vol. 276; 2001: 34631-34636. 6. Lee JH, Welch DR: Suppression of metastasis in human breast carcinoma MDA-MB-435 cells after transfection with the metastasis suppressor gene, KiSS-1. Cancer Res 1997, 57(12):2384-2387. 7. Muir AI, Chamberlain L, Elshourbagy NA, Michalovich D, Moore DJ, Calamari A, Szekeres PG, Sarau HM, Chambers JK, Murdock P et al: AXOR12, a novel human G protein-coupled receptor, activated by the peptide KiSS-1. J Biol Chem 2001, 276(31):28969-28975. 8. Ohtaki T, Shintani Y, Honda S, Matsumoto H, Hori A, Kanehashi K, Terao Y, Kumano S, Takatsu Y, Masuda Y et al: Metastasis suppressor gene KiSS-1 encodes peptide ligand of a G-protein-coupled receptor. Nature 2001, 411(6837):613-617. 9. Stathatos N, Bourdeau I, Espinosa AV, Saji M, Vasko VV, Burman KD, Stratakis CA, Ringel MD: KiSS-1/G protein-coupled receptor 54 metastasis suppressor pathway increases myocyte-enriched calcineurin interacting protein 1 expression and chronically inhibits  176 calcineurin activity. In: J Clin Endocrinol Metab. vol. 90; 2005: 5432- 5440. 10. Yan C, Wang H, Boyd DD: KiSS-1 represses 92-kDa type IV collagenase expression by down-regulating NF-kappa B binding to the promoter as a consequence of Ikappa Balpha -induced block of p65/p50 nuclear translocation. J Biol Chem 2001, 276(2):1164-1172. 11. Shoji S, Tang XY, Umemura S, Itoh J, Takekoshi S, Shima M, Usui Y, Nagata Y, Uchida T, Osamura RY et al: Metastin Inhibits Migration and Invasion of Renal Cell Carcinoma with Overexpression of Metastin Receptor. In: Eur Urol. 2008. 12. Bilban M, Ghaffari-Tabrizi N, Hintermann E, Bauer S, Molzer S, Zoratti C, Malli R, Sharabi A, Hiden U, Graier W et al: Kisspeptin-10, a KiSS- 1/metastin-derived decapeptide, is a physiological invasion inhibitor of primary human trophoblasts. J Cell Sci 2004, 117(Pt 8):1319-1328. 13. Yoshioka K, Ohno Y, Horiguchi Y, Ozu C, Namiki K, Tachibana M: Effects of a KiSS-1 peptide, a metastasis suppressor gene, on the invasive ability of renal cell carcinoma cells through a modulation of a matrix metalloproteinase 2 expression. In: Life Sci. 2008. 14. Navenot JM, Wang Z, Chopin M, Fujii N, Peiper SC: Kisspeptin-10- induced signaling of GPR54 negatively regulates chemotactic responses mediated by CXCR4: a potential mechanism for the metastasis suppressor activity of kisspeptins. Cancer Res 2005, 65(22):10450-10456. 15. Masui T, Doi R, Mori T, Toyoda E, Koizumi M, Kami K, Ito D, Peiper SC, Broach JR, Oishi S et al: Metastin and its variant forms suppress migration of pancreatic cancer cells. Biochem Biophys Res Commun 2004, 315(1):85-92. 16. Ringel MD, Hardy E, Bernet VJ, Burch HB, Schuppert F, Burman KD, Saji M: Metastin receptor is overexpressed in papillary thyroid cancer and activates MAP kinase in thyroid cancer cells. J Clin Endocrinol Metab 2002, 87(5):2399. 17. Teles MG, Bianco SD, Brito VN, Trarbach EB, Kuohung W, Xu S, Seminara SB, Mendonca BB, Kaiser UB, Latronico AC: A GPR54- activating mutation in a patient with central precocious puberty. In: N Engl J Med. vol. 358; 2008: 709-715. 18. Nash KT, Phadke PA, Navenot JM, Hurst DR, Accavitti-Loper MA, Sztul E, Vaidya KS, Frost AR, Kappes JC, Peiper SC et al: Requirement of KISS1 secretion for multiple organ metastasis suppression and  177 maintenance of tumor dormancy. J Natl Cancer Inst 2007, 99(4):309- 321. 19. DeWire SM, Ahn S, Lefkowitz RJ, Shenoy SK: Beta-arrestins and cell signaling. Annu Rev Physiol 2007, 69:483-510. 20. Moore CA, Milano SK, Benovic JL: Regulation of receptor trafficking by GRKs and arrestins. Annu Rev Physiol 2007, 69:451-482. 21. Seminara SB, Messager S, Chatzidaki EE, Thresher RR, Acierno JS, Jr., Shagoury JK, Bo-Abbas Y, Kuohung W, Schwinof KM, Hendrick AG et al: The GPR54 gene as a regulator of puberty. N Engl J Med 2003, 349(17):1614-1627. 22. Wacker JL, Feller DB, Tang XB, Defino MC, Namkung Y, Lyssand JS, Mhyre AJ, Tan X, Jensen JB, Hague C: Disease-causing Mutation in GPR54 Reveals the Importance of the Second Intracellular Loop for Class A G-protein-coupled Receptor Function. J Biol Chem 2008, 283(45):31068-31078. 23. Dhillo WS, Chaudhri OB, Thompson EL, Murphy KG, Patterson M, Ramachandran R, Nijher GK, Amber V, Kokkinos A, Donaldson M et al: Kisspeptin-54 stimulates gonadotropin release most potently during the preovulatory phase of the menstrual cycle in women. In: J Clin Endocrinol Metab. vol. 92; 2007: 3958-3966. 24. Semple RK, Achermann JC, Ellery J, Farooqi IS, Karet FE, Stanhope RG, O'Rahilly S, Aparicio SA: Two novel missense mutations in g protein- coupled receptor 54 in a patient with hypogonadotropic hypogonadism. J Clin Endocrinol Metab 2005, 90(3):1849-1855. 25. Goldsmith ZG, Dhanasekaran DN: G protein regulation of MAPK networks. Oncogene 2007, 26(22):3122-3142. 26. Gutkind JS: Regulation of mitogen-activated protein kinase signaling networks by G protein-coupled receptors. Sci STKE 2000, 2000(40):RE1. 27. Luttrell LM: Composition and function of g protein-coupled receptor signalsomes controlling mitogen-activated protein kinase activity. J Mol Neurosci 2005, 26(2-3):253-264. 28. Harms JF, Welch DR, Miele ME: KISS1 metastasis suppression and emergent pathways. Clin Exp Metastasis 2003, 20(1):11-18. 29. Nash KT, Welch DR: The KISS1 metastasis suppressor: mechanistic insights and clinical utility. Front Biosci 2006, 11:647-659.  178 30. Labrie F, Belanger A, Luu-The V, Labrie C, Simard J, Cusan L, Gomez J, Candas B: Gonadotropin-releasing hormone agonists in the treatment of prostate cancer. Endocr Rev 2005, 26(3):361-379. 31. Whistler JL, Gerber BO, Meng EC, Baranski TJ, von Zastrow M, Bourne HR: Constitutive activation and endocytosis of the complement factor 5a receptor: evidence for multiple activated conformations of a G protein-coupled receptor. Traffic 2002, 3(12):866-877. 32. Wilbanks AM, Laporte SA, Bohn LM, Barak LS, Caron MG: Apparent loss-of-function mutant GPCRs revealed as constitutively desensitized receptors. Biochemistry 2002, 41(40):11981-11989. 33. Arai AC, Xia YF, Suzuki E, Kessler M, Civelli O, Nothacker HP: Cancer metastasis-suppressing peptide metastin upregulates excitatory synaptic transmission in hippocampal dentate granule cells. J Neurophysiol 2005, 94(5):3648-3652. 34. Liu X, Lee K, Herbison AE: Kisspeptin excites gonadotropin-releasing hormone (GnRH) neurons through a phospholipase C / calcium- dependent pathway regulating multiple ion channels. In: Endocrinology. 2008.    179 6 DIFFERENTIAL TRANSCRIPTION IN THE HYPOTHALAMUS OF GPR54 AND KISS1 KNOCKOUT MICE REVEALED BY ONE MILLION EXON ARRAY ANALYSIS 5         Objective: To analyze differential gene expression between Gpr54 and Kiss1 knockout mice compared to wildtype animals to elucidate novel genes involved in the regulation of the ligand-receptor pair.          5  A version of this chapter will be submitted for publication. Prentice LM, d’Anglemont de Tassigny X, McKinney S, Fee J, Ruiz de Algara T, Huntsman DG, Colledge WH, and Aparicio SAJ. Differential transcription in the hypothalamus of Gpr54 and Kiss1 knockout mice revealed by one million exon array analysis.  180 6.1 Introduction In 2003 it was discovered that the kisspeptin-GPR54 ligand-receptor pair are significant players in the endocrine regulation of mammalian reproduction [1, 2]. It has been established subsequently that Kp binding to GPR54 releases gonadotropin releasing hormone (GnRH) from the hypothalamus to stimulate the pituitary-gonadal axis in a multitude of species including humans [3-15]. In rodents, Kp is expressed in the arcuate nucleus (ARC) of the hypothalamus and also in the rostral periventricular area of the 3rd ventricle (RP3V) which includes the anteroventral periventricular nucleus (AVPV). Rising estrogen levels have a positive feedback effect on Kp expression in the AVPV to initiate the LH surge required for ovulation in females [16-19]. Conversely, estrogen and testosterone have a negative feedback effect on Kp expression in the ARC that is regulated through estrogen receptor alpha (ER) and also through the androgen receptor (AR) in males [16, 18-21].  What remains to be determined are the mechanisms controlling this sex steroid regulation of Kiss1 and the consequences of loss of Kp signalling on downstream expression in the hypothalamus. We have generated Gpr54 and Kiss1 knockout mice that model the hypogonadotropic hypogonadism (HH) found in humans with GPR54 mutations [2, 10, 22]. In order to assess what other genes may be affected in the hypothalamus, we compared gene expression between the two knockout genotypes as compared to wildtype mice of the same parental strain.  181 Affymetrix Exon 1.0 ST Array analysis of Gpr54 and Kiss1 KO mice hypothalami identified a novel gene list of possible regulators of Gpr54 and Kiss1 expression as well as genes potentially regulated by Gpr54 and Kiss1. QPCR validation confirmed the novel gene list determined by the Affymetrix analysis and supported the current literature that GPR54/Kp are partially regulated through an endocrine feedback loop.  6.2 Material and Methods 6.2.1 Experimental procedure i) To discover novel gene candidates that may be involved in the Kp-GPR54 signalling pathway, we assessed gene expression differences in the hypothalamus of Kiss1 and Gpr54 knockout mice (KKO, GKO) compared to wild- type mice (WT). An Affymetrix Exon 1.0 ST Array was used to assess gene expression initially, and these results were validated using real-time PCR and the Applied Biosystems Low Density Array (LDA). ii) To account for hormonally regulated genes that may be differentially regulated as a consequence of sexual immaturity yet not directly affected by Kp and/or GPR54, a hormonally controlled group of mice were assessed. To ensure equal hormone exposure in all genotypes (GKO, KKO, WT), all mice were castrated prior to treatment. Treatment consisted of either a testosterone implant or an empty sialistic control. The hypothalamus was again isolated for assessment of differential gene expression, this time using a smaller LDA. The genes chosen for the smaller 48 gene LDA were based on the results of the previous larger 95  182 gene LDA that had been originally designed from the Affymetrix results. The experimental procedure is summarized in Figure 6.1.  6.2.2 Animals Gpr54 and Kiss1 knockout mice have been previously described [2, 10, 22]. Male 129S6/Sv/Ev wild-type, 129S6/Sv/Ev Gpr54- or 129S6/Sv/Ev Kiss1- knockout mice were housed under conditions of 12 hours of light with ad libitum access to food and water. The average age of the mice from the first analysis was 60-70 days and 90 days for the second hormonally controlled group. All experimental protocols were performed under the authority of a United Kingdom Home Office Project License and were approved by the Cambridge Animal Ethics Committee.  6.2.3 Castration and testosterone implants Adult males were bilaterally castrated under general anaesthesia using Ketamin/Xyline. Castrated mice were divided into two groups: bilateral castration plus empty implant or bilateral castration plus testosterone implant. Testosterone implants were manually and aseptically prepared in the laboratory using silicone tubing (0.058 inch ID / 0.077 inch OD; Dow Corning) filled with crystalline testosterone (T-1500; Sigma Aldrich, UK), and sealed with adhesive silicone type A glue [23]. Implants were inserted subcutaneously at the time of castration. Mice were allowed approximately 3 - 4 weeks for recovery (the first week with paracetamol in the water supply), and killed by CO2 exposure. Blood was collected in a heparinized syringe from the inferior vena cava and centrifuged at  183 1,000x g for 10 min at 4°C. The plasma supernatant samples were collected and stored at -20°C.  6.2.4 Testosterone assay Free testosterone was measured by using an ELISA kit (DB52181; IBL, Hamburg, Germany) with a sensitivity of 0.17 pg/ml and intra- and inter-assay variation coefficients respectively 8.9% and 8.8% [22].  6.2.5 Hypothalamus extraction After removal of the brain, the meninges and optic chiasm were discarded and the hypothalamus was isolated. The external limits for this dissection are: lateral, the external border of the medial preoptic area and more caudally, the lateral borders of the mammillary nucleus; dorsal, 1.5 mm depth; anterior, the anterior limit of the nucleus of the vertical limb of the diagonal band (bregma + 1.34 mm); and posterior, the posterior limit of the mammillary nucleus (bregma – 3.40 mm). The whole hypothalamus contained at least the following main hypothalamic nuclei: anteroventral periventricular nucleus, anterodorsal preoptic nucleus, anterior-hypothalamic nucleus, magnocellular lateral hypothalamic nucleus, supraoptic and paraventricular nuclei, medial preoptic area and the nucleus of the vertical limb of the diagonal band. Immediately after dissection, hypothalami samples were collected in 600 l of RNAlater (Applied Biosystems, UK) and kept at 4°C for 24 h and then stored at -20°C until RNA extraction.   184   Figure 6.1 Summary of the experimental procedure Initially, gene expression was analyzed using the Affymetrix Exon 1.0 ST Array and the results from this analysis were validated using the ABI Low Density Array (LDA) covering 70 genes from the Affymetrix analysis. Both platforms used RNA from mouse hypothalami from the three different genotypes: Kiss1 knockout mice (KKO), Gpr54 knockout mice (GKO), and wildtype mice (WT). Based on the results from the larger LDA array, a second smaller LDA was used to assess hormonally controlled genes. The mice for this experiment were all castrated and either treated with testosterone implant or an empty control prior to hypothalamus extraction. There were at least three biological replicates for each group.  185  6.2.6 RNA extraction RNA was extracted using the Qiazol method (Invitrogen, Carlsbad, CA) that is recommended for fatty tissues. Whole hypothalami were removed from RNAlater, with excess reagent blotted off of the tissue with a kimwipe before being placed in 100-200 μl of Qiazol. The hypothalami were then homogenized using a Kontes Pellet Pestle (Fischer Scientific, Ottawa, ON) hand-held homogenizer until a uniform mixture was achieved. The remaining Qiazol up to 1 ml was added to the homogenized mixture and the total homogenate was placed in a phase lock tube to separate out the aqueous phase through centrifugation. RNA was precipitated out of the aqueous phase with an equal volume of isopropanol and pelletted by centrifugation. Seventy percent ethanol was used to wash the pellet that was then air-dried, resuspended into 5.5-11.5 μl of RNase/DNase free water, and heated to 65°C for 10 min. One and a half microliters of RNA was set aside for Agilent analysis and RNA was stored at -80°C.  6.2.7 Agilent analysis RNA concentration and integrity (RIN) were determined by the Agilent 2100 bioanalyzer as per manufacturer’s recommendations. Briefly, 1 μl of heat denatured RNA mixed with 5 μl of Nano Marker was run on a RNA 6000 NanoChip (Agilent Technologies, Mississauga, ON) already prepped with pre- filtered Nano gel matrix mixed with Nano dye concentrate. RNA samples were compared to 1 μl of NanoChip RNA 6000 ladder.  186  6.2.8 Affymetrix procedure Hypothalamic RNA was prepared using the GeneChip Whole Transcript (WT) Sense Target (ST) Labeling Assay (Affymetrix, Santa Clara, CA) as per manufacturer’s recommendations before being hybridized to a GeneChip Mouse Exon 1.0 ST Array (Affymetrix, Santa Clara, CA). One microgram of RNA was used as starting material with all samples having a RIN ranging between 8.2-9.0. Starting RNA was mixed with Control Poly-A-RNA before using the RiboMinus Transcriptome Isolation Kit (Invitrogen, Carlsbad, CA) as per Affymetrix recommendations to reduce the rRNA contamination within the sample. Each batch of RNA run through the RiboMinus kit was run in parallel with mouse liver RNA that had either been rRNA reduced or left unreduced as a comparison for effectiveness of the RiboMinus assay. The rRNA reduced samples and liver controls were then purified and concentrated using the GeneChip In Vitro Transcription (IVT) cRNA Cleanup Kit and assessed by Agilent analysis. Successfully rRNA reduced samples continued forward to first strand cDNA synthesis using T7-(N)6 primers and SuperScript II enzyme before second strand cDNA synthesis using DNA Polymerase I. Within 10 min of second strand cDNA synthesis, samples were run through the GeneChip WT cDNA Amplification Kit before IVT cRNA Cleanup and then assessed for cRNA concentration by NanoDrop analysis (NanoDrop Technologies, Wilmington, DE). Samples with cRNA concentrations equal to or greater than 10 μg in a total volume of 6.5 μl were carried forward to the second cycle of first-strand cDNA synthesis using the  187 GeneChip WT cDNA synthesis Kit. Random primers with a deoxyuridine triphosphate (dUTP) component mixed within the deoxynucleotide triphosphates (dNTPs) were used. The single stranded cDNA was then treated with RNase H to hydrolyze any remaining cRNA and run through the cDNA Cleanup Spin Columns as per the GeneChip Sample Cleanup Module. Eluted cDNA was assessed by NanoDrop for sample concentration. Finally, 5.5 μg of single stranded cDNA was fragmented with Uracil DNA Glycosylase (UDG) and APE 1 then labeled with biotin allonamide triphosphate (TdT) using the GeneChip WT Terminal Labeling Kit before being hybridized for 17 h to the Mouse 1.0 ST Array chip in the GeneChip Hybridization Oven 640 using the GeneChip Hybridization, Wash and Stain Kit. Before scanning the chip on the GeneChip Scanner 3000 7G, each chip was run through the GeneChip Fluidics Station 450 for washing and staining with Streptavidin Phycoerythrin (SAPE). When the sample yielded a concentration less than required for subsequent steps in the protocol, the process was started over again and resulting volumes amalgamated (and in some instances vacuum centrifuged to increase the concentration) before moving forward to the next step. The protocol was also attempted with 2 μg of RNA as starting material as advised by technical support but an increase in yield was not achieved.  6.2.9 Quantitative real-time polymerase chain reaction (QPCR) The Applied Biosystems (ABI) 7900HT Fast Real-Time System was used to amplify and detect mouse cDNA transcripts (Applied Biosystems, Foster City,  188 CA). For high throughput detection a Taqman Low Density Array (LDA) Card from ABI consisting of 95 genes including two endogenous control genes (GAPDH and 18S rRNA) was custom ordered. To assess the second group of mice independent of hormonal feedback, a second smaller LDA set was ordered consisting of 48 genes. The gene list was similar to that of the first larger LDA and had the same two endogenous controls. The complete list of genes used for both LDAs are in Tables 6.1 and 6.2.  6.2.10 Statistical analysis Stratagene ArrayAssist version 5.1.0 was used to analyzed the Affymetrix data and create volcano plots. Affymetrix chip data was quantile normalized and corrected using the GC-RMA algorithm [24].  Experimental groups were compared using unpaired t-tests. QPCR data was analyzed using linear mixed effects models, with experimental groups compared via the likelihood ratio chi- square statistic [25, 26].  Adjustment for multiple comparisons was performed using the Benjamini-Hochberg method [27].  For the testosterone challenge experiment, a linear mixed effects model was used to relate raw Ct values from loading control genes (Gapdh, 18S RNA) and test genes. Although all factors could have been put in one 2x2x3 model, for simplicity the data from Kiss1 and Gpr54 knockouts was modeled separately, in two 2x2x2 factorial designs (genotype: wt or ko; treatment: T- or T+; gene: test or loading control). The relationship between the model-estimated means for each of the conditions allowed inference of four main effects (Figure 6.2).  189 Table 6.1 Gene list for the 96 Low Density Array  96 Low Density Array* Gene Assay ID  Gene Assay ID Gene Assay ID 18S Hs99999901_s1 Htr2c Mm00434127_m1 Pdyn Mm00457572_m1 Abca8a Mm00462440_m1 Hyal2 Mm00477731_m1 Pgm1 Mm00804141_m1 Acsm3 Mm00489774_m1 Il15ra Mm00500457_m1 Pgr Mm00435625_m1 Adamtsl4 Mm00523242_m1 Il8ra Mm00731329_s1 Phtf2 Mm00557065_m1 Ar Mm00442688_m1 Irak3 Mm00518541_m1 Ppef2 Mm00448366_m1 Boc Mm00552900_m1 Itgax Mm00498698_m1 Prickle3 Mm00558283_m1 Centd2 Mm00546699_g1 Itgbl1 Mm00520942_m1 Pus7 Mm00617871_m1 Cln3 Mm00487021_m1 Itpr3 Mm00446540_m1 Rab34 Mm00446343_g1 Col7a1 Mm00483818_m1 Kiss1 Mm00617576_m1 Rragd Mm00546741_m1 Ddx3y Mm00465349_m1 Kiss1r Mm00475046_m1 Sh3tc2 Mm00553970_m1 Dmn Mm00809202_s1 Krit1 Mm00459502_m1 Six2 Mm00807058_m1 Dmrt3 Mm00616649_m1 Lep Mm00434759_m1 Slc25a25 Mm00525104_m1 Eda Mm00438653_m1 Lepr Mm00440174_m1 Slc26a8 Mm00524836_m1 Edc4 Mm00725090_m1 Lhb Mm00656868_g1 Sp1 Mm00489039_m1 Eif2s3y Mm00468995_g1 Lhcgr Mm00442931_m1 Speer4b Mm00835620_m1 Elk3 Mm00469054_m1 Lrdd Mm00502614_m1 Sphk1 Mm01252544_m1 Elmo3 Mm00555221_g1 Mapk1 Mm00442479_m1 Srpk3 Mm00444746_m1 Ermap Mm00469273_m1 Med23 Mm00518410_m1 Srrm1 Mm00489728_m1 Ero1l Mm00469296_m1 Metapl1 Mm00491809_m1 Sult1c2 Mm00471845_m1 Esr1 Mm00433149_m1 Mmp2 Mm00439506_m1 Svep1 Mm00465696_m1 Esr2 Mm00599819_m1 Mmp28 Mm00712992_m1 Tac2 Mm00436885_m1 Fsd2 Mm00556634_m1 Mmp9 Mm00442991_m1 Tacr3 Mm00445346_m1 Fzd10 Mm00558396_s1 Mobkl2c Mm00774347_m1 Taf12 Mm00499416_m1 Gapdh Mm99999915_g1 Myh9 Mm00502575_m1 Tcf7l2 Mm00501505_m1 Glis3 Mm00615386_m1 Ngef Mm00451232_m1 Tec Mm00443230_m1 Gnrhr Mm00439143_m1 Npas4 Mm00463644_m1 Tmem144 Mm00510477_m1 Golt1a Mm00503319_m1 Npy Mm00445771_m1 Txnip Mm00452393_m1 Gpr143 Mm00440553_m1 Nr2f2 Mm00772789_m1 Ucn Mm00445261_m1 Gpr146 Mm01700739_m1 Oas3 Mm00460944_m1 Wnt5a Mm00437347_m1 Gtf3a Mm00550608_m1 Olig2 Mm01210556_m1 Zfp472 Mm00461969_m1 Hapln2 Mm00480745_m1 Opn4 Mm00443523_m1 Zmym6 Mm00624222_m1 Hhip Mm00469580_m1 Pbx2 Mm00479560_m1 *One position is empty on the part of the manufacturer       190 Table 6.2 Gene list for the 48 Low Density Array  48 Low Density Array Gene Assay ID Gene Assay ID 18S Hs99999901_s1 Klk1b22 Mm02343755_g1 Abca8a Mm00462440_m1 Krit1 Mm00459502_m1 Acsm3 Mm00489774_m1 Lhcgr Mm00442931_m1 Ar Mm00442688_m1 Lrdd Mm00502614_m1 Arap1 Mm00546699_g1 Mmp2 Mm00439508_m1 Boc Mm00552900_m1 Mmp28 Mm00712992_m1 Ddx3y Mm00465349_m1 Mmp9 Mm00442991_m1 Eif2s3y Mm00468995_g1 Mobkl2c Mm00774347_m1 Elk3 Mm00469054_m1 Ngef Mm00451232_m1 Ero1l Mm00469296_m1 Npas4 Mm00463644_m1 Esr1 Mm00433149_m1 Nr2f2 Mm00772789_m1 Esr2 Mm00599819_m1 Olig2 Mm01210556_m1 Fzd10 Mm00558396_s1 Pgm1 Mm00804141_m1 Gapdh Mm99999915_g1 Pgr Mm00435625_m1 Glis3 Mm00615386_m1 Phtf2 Mm00557065_m1 Gnrhr Mm00439143_m1 Six2 Mm00807058_m1 Hapln2 Mm00480745_m1 Synm Mm00809202_s1 Hhip Mm00469580_m1 Tac2 Mm00436885_m1 Htr2c Mm00434127_m1 Taf12 Mm00499416_m1 Hyal2 Mm00477731_m1 Tcf7l2 Mm00501505_m1 Il15ra Mm00500457_m1 Tec Mm00443230_m1 Itgax Mm00498698_m1 Tmem144 Mm00510477_m1 Kiss1 Mm00617576_m1 Txnip Mm00452393_m1 Kiss1r Mm00475046_m1 Wnt5a Mm00437347_m1 Dmn=Synm, Centd2=Arap1  191   Figure 6.2 Summary of statistical grouping of the hormonally controlled LDA assay A) Results were deemed to be a genotype only effect if the measured fold change between the wildtype (WT) and knockout (KO) mice was the same in both of the hormone T+ and T- conditions. B) Similarly, result is a hormone only effect if the measured fold change between the T+ and T- conditions was the same for both of the genotype WT and KO conditions. C) Genes were classified as having both a genotype and hormone effect when there was a proportional difference between the two comparisons. D) Genes were classified as having a genotype and hormone effects with interaction when there was a disproportionate difference between the two comparisons that was not equal to 1.  192  These were: genotype only transcription differences, hormone treatment only transcription differences, hormone and genotype differences where the rate of difference is symmetrical (no interaction between effects), and hormone and genotype differences where the differences between groups show an effect interaction (eg the difference for hormone treatment is disproportionately greater in one genotype). The linear model was fitted using the lmer function from the lme4 package implemented in R-2.7.0 [28, 29]. The model output reports the estimated mean, 95% confidence intervals, relative effect and direction, and p- value (adjusted for multiple corrections by the Benjamini Hochberg method).  6.3 Results 6.3.1 Microarray identification of candidate hypothalamic deregulated transcripts from GPR54 and kisspeptin knockout mice. We compared quantile normalized probe intensity values from Affymetrix whole mouse exon array chips where hybridization was performed with wildtype, Gpr54 knockout (GKO) or Kiss1 knockout (KKO) hypothalamic RNA. Affymetrix results were compared between genotypes, specifically gene expression of all wildtype mice were grouped together and compared with all knockout mice grouped together (WT vs KO) or with Kiss1 knockout mice alone (WT vs KKO) or Gpr54 knockout mice alone (WT vs GKO). Additionally, Gpr54 knockout mice were compared with Kiss1 knockout mice (GKO vs KKO) to give a total of four comparative groups. Gene level and probe level summarization was used in the  193 comparisons and from each of these we selected gene loci showing p<0.05 and expression fold differences of 1.5 or greater, as candidates for further analysis (yellow highlighted regions in the volcano plots shown in Figure 6.2).  This analysis yielded 213 gene loci, putatively exhibiting differential transcription (Figure 6.3). To validate the initial array based measurements of transcription, we selected 70 candidate loci for further validation by QPCR. The selections were prioritized on the strength of expression, consistency of differences between knockouts, known or suspected roles in hormonal regulation, and the availability of quenching QPCR probes for low density 384 well plate QPCR plate analysis. Within these criteria, all transcription factors, transmembrane receptors, and signalling molecules that were available were included. QPCR sets for 18S RNA and Gapdh were included in each array as loading controls. Finally, we also selected a number of genes of interest based on known role in gonadotropic signalling, that were not differentially expressed by microarray analysis. This resulted in 95 gene loci, of which 25 were controls or genes of interest and 70 were genes selected on the basis of differential expression by array analysis (Table 6.1).  We used the LDA QPCR array to assay each gene locus at least 3 times per sample (technical replicates) and over 3 biological replicates of RNA prepared from microdissected hypothalamic tissue from wildtype, GKO and KKO mice. After analysis of the CT values by a linear mixed effects model, 18 loci showed statistically significant differential expression in one of the groups (Table 6.3) and were carried forward for further analysis.  194    Figure 6.3 Volcano plots from the Affymetrix Exon 1.0 ST Array Genes that were considered for further analysis are represented by yellow squares. The x-axis is the fold change for comparison groups that are indicated on the left side of the figures. The y-axis is the p-value among biological replicates.  195   Figure 6.4 Summary of Affymetrix results The pie chart represents the breakdown of the 213 unique genes by function based on the ExPASy proteomics server (http://www.expasy.ch).  196 Table 6.3 Genes with significant fold change from the 95 LDA assay   GKO vs WT KKO vs WT GeneTarget FC (95%LCI-UCI) Direction FC (95%LCI-UCI) Direction Abca8a 1.85 (1.33-2.59) Up 1.97 (1.33-2.90) Up Esr1 1.62 (1.37-1.92) Up 1.54 (1.32-1.78) Up Htr2c 1.34 (1.14-1.58) Up 1.41 (1.21-1.64) Up Kiss1 14.03 (10.86-18.11) Up NA NA Mmp2 1.51 (1.18-1.94) Up n.s. n.s. Npas4 2.60 (2.09-3.25) Up n.s. n.s. Boc n.s. n.s. 1.38 (1.14-1.67) Up Eif2s3y n.s. n.s. 1.26 (1.11-1.43) Up Ero1l n.s. n.s. 1.23 (1.07-1.41) Up Hapln2 n.s. n.s. 1.20 (1.05-1.37) Up Hhip n.s. n.s. 1.34 (1.11-1.63) Up Krit1 n.s. n.s. 1.21 (1.05-1.39) Up Lrdd n.s. n.s. 1.80 (1.29-2.51) Up Ngef n.s. n.s. 1.22 (1.07-1.40) Up Pgm1 n.s. n.s. 1.26 (1.07-1.48) Up Taf12 n.s. n.s. 1.31 (1.14-1.50) Up Tmem144 n.s. n.s. 2.61 (2.21-3.08) Up Txnip n.s. n.s. 1.36 (1.13-1.65) Up   197 6.3.2 Dissection of hormone dependent and independent hypothalamic gene transcription in Gpr54 and Kiss1 knockout mice. Expression analysis of intact GKO and KKO hypothalamus RNA revealed significant differential transcription. However, since GKO and KKO mice fail to undergo puberty an unknown number of these transcriptional differences could be explained by the hormonal milieu. To address this, we asked which of the gene loci exhibited hormonally responsive transcription, by measuring transcriptional differences in the presence or absence of testosterone (T). WT, GKO and KKO male mice were castrated and implanted with either a sham silastic implant, or a T containing implant. After 4 weeks exposure, the mice were sacrificed, the hypothalamus microdissected, and the RNA extracted. To confirm exposure to T, we measured serum testosterone (Table 6.4). Each gene locus was assayed with at least 4 technical replicates and 3 biological replicates. To reduce the chance genotype or hormonal variance of loading controls, we used two independent loading controls, 18S RNA and Gapdh. The data were analysed separately using a linear mixed effects model (methods). We analyzed GKO and KKO mice separately in a 2x2x2 design, considering gene (test, loading control), treatment (T+, T-) and genotype (WT, GKO, KKO) as categorical variables. Tables 6.5-6.12 summarize the statistically significant (p<0.05) model results for hormone effect, genotype effect, hormone+genotype no interaction and hormone+genotype with interaction, for the GKO and KKO mice, separately.   198 Table 6.4 Characteristics of hormonally treated mice  Mouse ID Genotype Sex Weight Testosterone Implant Date of Birth Date of Castration Date of Sacrifice KJ74 K-/- Male 20 g 0 pg/mL Empty 29/08/08 15/10/08 12/11/08 KJ59 K-/- Male 21 g 0 pg/mL Empty 17/08/08 15/10/08 12/11/08 KJ10 K-/- Male 20 g 0 pg/mL Empty 04/08/08 30/09/08 21/10/08 KJ25 K-/- Male 20 g 5.7 pg/mL Testosterone 25/07/08 30/09/08 21/10/08 KJ55 K-/- Male 21 g 2.8 pg/mL Testosterone 15/08/08 15/10/08 12/11/08 KJ47 K-/- Male 22 g 3.4 pg/mL Testosterone 14/08/08 15/10/08 12/11/08 HL73 G-/- Male 21 g 0 pg/mL Empty 18/08/08 15/10/08 12/11/08 HL74 G-/- Male 21 g 0 pg/mL Empty 18/08/08 15/10/08 12/11/08 HL27 G-/- Male 20 g 0 pg/mL Empty 19/07/08 30/09/08 21/10/08 HL61 G-/- Male 17 g 5.1 pg/mL Testosterone 24/08/08 15/10/08 12/11/08 HK97 G-/- Male 23 g 8.8 pg/mL Testosterone 07/07/08 30/09/08 21/10/08 HL49 G-/- Male 22 g 3.6 pg/mL Testosterone 25/07/08 30/09/08 21/10/08 KJ52 WT+/+ Male 21 g 0 pg/mL Empty 14/08/08 15/10/08 12/11/08 KJ64 WT+/+ Male 29 g 0 pg/mL Empty 19/08/08 15/10/08 12/11/08 HL31 WT+/+ Male 28 g 0 pg/mL Empty 19/07/08 30/09/08 21/10/08 HL30 WT+/+ Male 26 g 4.2 pg/mL Testosterone 19/07/08 30/09/08 21/10/08 HK99 WT+/+ Male 27 g 4.8 pg/mL Testosterone 07/07/08 30/09/08 21/10/08 KJ46 WT+/+ Male 24 g 7 pg/mL Testosterone 14/08/08 15/10/08 12/11/08  Table 6.5 KKO hormone only effect genes compared with testosterone treatment  KKO Hormonal Effect Gene Target FC (95%CI) Direction p-value Esr1 1.23(1.14-1.32) Down 0.0003 Gnrhr 2.33(1.63-3.33) Down 0.0009  Table 6.6 GKO hormone only effect genes compared with testosterone treatment  GKO Hormonal Effect Gene Target FC (95%CI) Direction p-value Abca8a 1.57(1.33-1.87) Down 2.83E-05 Hyal2 1.13(1.05-1.22) Down 0.0422 Klk1b22 2.16(1.43-3.25) Down 0.0042 Mmp2 1.23(1.10-1.37) Down 0.0113 Pgr 1.28(1.18-1.37) Up 5.66E-07  199 Table 6.7 KKO genotype effect only genes compared with WT  KKO Genotype Effect Gene Target FC (95%CI) Direction p-value Ar 1.16(1.08-1.25) Down 0.0272 Eif2s3y 1.27(1.09-1.48) Down 0.0225 Tmem144 1.50(1.30-1.73) Up 6.12E-06  Table 6.8 GKO genotype effect only genes compared with WT  GKO Genotype Effect Gene Target FC (95%CI) Direction p-value Arap1 1.21(1.10-1.34) Down 0.0042 Boc 1.24(1.11-1.39) Down 0.0136 Eif2s3y 1.48(1.25-1.74) Down 0.0003 Ero1l 1.27(1.18-1.38) Down 2.61E-05 Esr2 1.63(1.46-1.82) Down 4.10E-10 Glis3 1.32(1.18-1.48) Down 0.0004 Hapln2 1.43(1.28-1.60) Down 2.61E-05 Hhip 1.63(1.46-1.82) Down 4.68E-10 Htr2c 1.24(1.13-1.36) Down 0.0003 Hyal2 1.25(1.13-1.39) Down 0.0004 Lhcgr 1.61(1.30-1.99) Down 0.0042 Lrdd 1.47(1.28-1.68) Down 3.88E-05 Mmp28 1.48(1.25-1.73) Down 0.0023 Mobkl2c 1.36(1.22-1.53) Down 0.0004 Npas4 1.65(1.45-1.87) Down 2.06E-08 Nr2f2 1.28(1.18-1.39) Down 2.46E-05 Olig2 1.48(1.32-1.65) Down 8.34E-07 Pgm1 1.26(1.15-1.37) Down 0.0003 Phtf2 1.25(1.15-1.36) Down 0.0001 Taf12 1.28(1.17-1.39) Down 1.57E-05 Wnt5a 1.36(1.24-1.50) Down 1.39E-06    200 Table 6.9 KKO genes with independent genotype and hormone effect  KKO Genotype Effect Hormonal Effect Gene Target FC (95%CI) Direction FC (95%CI) Direction p-value Abca8a 1.21(1.06-1.37) Up 1.64(1.45-1.86) Down 1.75E-09 Ddx3y 1.18(1.06-1.32) Down 1.25(1.12-1.39) Down 0.0013 Fzd10 1.16(1.03-1.31) Up 1.18(1.05-1.33) Down 0.0272   Table 6.10 GKO genes with independent genotype and hormone effect  GKO Genotype Effect Hormonal Effect Gene Target FC (95%CI) Direction FC (95%CI) Direction p-value Abca8a 1.35(1.15-1.58) Down 1.58(1.35-1.85) Down 1.39E-07 Ddx3y 1.52(1.31-1.77) Down 1.21(1.04-1.40) Down 1.52E-05 Esr1 1.13(1.04-1.22) Up 1.40(1.30-1.52) Down 5.45E-10 Fzd10 1.36(1.20-1.53) Down 1.16(1.03-1.31) Down 6.00E-05 Kiss1 1.96(1.64-2.35) Up 12.15(10.15-14.55) Down 4.29E-46 Mmp2 1.41(1.27-1.56) Down 1.23(1.12-1.36) Down 2.06E-08 Pgr 1.28(1.18-1.38) Down 1.27(1.18-1.37) Down 1.51E-10 Tmem144 1.86(1.60-2.16) Down 1.28(1.10-1.48) Down 1.51E-10   Table 6.11 KKO hormone and genotype effect interacting genes   Genotype Effect within Hormonal Group Hormonal Effect within Genotype KKO No testosterone Testosterone Wildtype Knockout Gene Target FC (95%CI) Dir FC (95%CI) Dir FC (95%CI) Dir FC (95%CI) Dir p-value Acsm3 1.23(1.03- 1.48) Up 1.27(1.03- 1.56) Down 1.86(1.53- 2.26) Up 1.19(0.98- 1.45) Up 9.80E-06 Klk1b22 1.30(0.92- 1.84) Down 4.30(3.01- 6.15) Down 1.34(0.97- 1.84) Down 4.43(3.01- 6.51) Down 1.75E-09 Mmp9 1.03(0.68- 1.56) Down 3.75(2.44- 5.75) Down 1.08(0.74- 1.59) Down 3.93(2.48- 6.22) Down 0.0127 Six2 1.08(0.84- 1.40) Up 1.99(1.54- 2.57) Up 1.60(1.24- 2.07) Down 1.15(0.89- 1.49) Up 1.14E-05 Tec 1.07(0.86- 1.32) Down 1.72(1.39- 2.12) Up 1.63(1.32- 2.02) Down 1.12(0.91- 1.38) Up 1.82E-05 Txnip 1.16(1.00- 1.34) Down 1.22(1.06- 1.41) Up 1.66(1.44- 1.92) Down 1.18(1.02- 1.36) Down 3.01E-08     201  Table 6.12 GKO hormone and genotype effect interacting genes   Genotype Effect within Hormonal Group Hormonal Effect within Genotype GKO No testosterone Testosterone Wildtype Knockout Gene Target FC (95%CI) Dir FC (95%CI) Dir FC (95%CI) Dir FC (95%CI) Dir p-value Acsm3 1.26(1.01- 1.57) Up 2.28(1.84- 2.83) Down 1.93(1.54- 2.42) Up 1.49(1.21- 1.84) Down 2.02E-08 Ar 1.23(1.09- 1.38) Down 1.56(1.39- 1.75) Down 1.13(1.01- 1.27) Up 1.12(1.00- 1.25) Down 1.02E-08 Ddx3y 1.30(1.13- 1.50) Down 1.01(0.88- 1.16) Up 1.38(1.20- 1.59) Down 1.05(0.91- 1.20) Down 0.0004 Eif2s3y 1.33(1.10- 1.61) Down 1.10(0.91- 1.33) Up 1.40(1.16- 1.70) Down 1.05(0.87- 1.27) Up 0.0196 Elk3 1.47(1.29- 1.66) Down 1.15(1.02- 1.31) Down 1.09(0.96- 1.23) Down 1.17(1.03- 1.32) Up 2.88E-06 Esr1 1.08(0.96- 1.21) Down 1.31(1.17- 1.47) Down 1.28(1.14- 1.43) Down 1.55(1.39- 1.74) Down 1.36E-11 Fzd10 1.20(1.01- 1.42) Down 1.17(0.99- 1.40) Up 1.37(1.15- 1.63) Down 1.02(0.86- 1.22) Up 0.0315 Gnrhr 1.28(0.78- 2.12) Down 3.20(1.70- 6.03) Up 2.29(1.37- 3.82) Down 1.80(0.96- 3.35) Up 1.80E-03 Kiss1 1.95(1.58- 2.42) Up 1.09(0.88- 1.35) Up 9.12(7.36- 11.29) Down 16.34(13.19- 20.23) Down 5.71E-52 Klk1b22 1.25(0.71- 2.20) Up 2.70(1.61- 4.52) Down 1.08(0.61- 1.91) Down 3.65(2.17- 6.12) Down 0.0004 Krit1 1.10(0.94- 1.28) Down 1.48(1.27- 1.73) Down 1.06(0.91- 1.24) Up 1.27(1.09- 1.48) Down 0.0005 Mmp9 1.11(0.91- 1.35) Up 1.70(1.40- 2.07) Up 1.11(0.92- 1.35) Up 1.71(1.41- 2.08) Up 2.69E-07 Ngef 1.09(0.97- 1.23) Down 1.44(1.27- 1.62) Down 1.15(1.01- 1.29) Up 1.15(1.02- 1.30) Down 5.16E-05 Six2 1.05(0.73- 1.50) Down 2.22(1.55- 3.18) Up 1.78(1.24- 2.55) Down 1.30(0.91- 1.87) Up 0.0022 Tec 2.48(1.95- 3.14) Down 1.75(1.38- 2.22) Down 1.63(1.29- 2.07) Down 1.16(0.91- 1.46) Down 2.03E-13 Txnip 1.04(0.80- 1.34) Down 1.81(1.40- 2.35) Up 1.85(1.43- 2.39) Down 1.02(0.79- 1.32) Up 8.79E-05     202    Figure 6.5 Summary of gene expression differences with a fold change 1.5 Each quadrant contains the gene list for both the Gpr54 knockout mice (GKO) and the Kiss1 knockout mice (KKO) that had a fold change difference of  1.5 in at least one of the comparisons. The quadrants are labeled by gene expression effect as determined by the hormonally controlled experiment.  203 The strongest effect is in the Kiss1 locus, measured in GKO mice which showed strong hormonal regulation (approximately 12 fold downregulated with T) and moderate genotype dependence (approximately 2 fold upregulated). With the 18S loading control, the regulation shows a weak interaction whereas with the Gapdh loading control, hormonal regulation and genotype regulation vary symmetrically and thus no interaction is reported. The next largest effects are for the locus Klk1b22 (also known as NGF peptidase) and the GnRH receptor (Gnrhr) both of which show a strong hormonal effect and a weak genotype effect but with some interaction (the hormone effect is relatively stronger in the KO than the WT mice), however with large variances.  Fewer statistically significant effects were seen in the KKO mice, although some loci (eg Klk1b22, Gnrhr, Tmem144) were regulated in common with GKO mice, albeit with effects of different magnitude. Many of the statistically significant results are of small absolute magnitude and considering the variances of the loading controls between genotypes and treatments, interpretation of differences of less than 1.5 fold may not be meaningful.  Thus considering effects of 1.5 fold or greater we observed the following: For Kiss1 knockout mice, only Gnrhr showed a pure hormonal effect, only Tmem144 showed a purely genotype driven change. Among the genotype and hormone variant changes, Klk1b22 showed the greatest differences, more than 2 fold, with testosterone exposure and this effect was greater in the knockout mice than the wildtypes (ie there is a statistically significant interaction).   204  For Gpr54 knockout mice, Klk1b22 showed a hormone only effect (downregulation) with the Gapdh control, but a hormone effect with genotype interaction using the 18S loading control. The direction and magnitude is similar to that observed in the KKO mice. Gnrhr shows a strong hormonal effect with a genotype interaction – downregulation in the wildtype mice and upregulation in the knockout mice. Tmem144 shows a strong genotype effect in the GKO mice (downregulation), as with KKO mice, but with an additional marginal hormone effect without genotype interaction. Genotype only effects were seen at > 1.5 fold but less than 2 fold for ER (Esr2), Hhip, Lhcgr, and Npas4.  We also noticed several matrix metalloproteinase (MMP) family members (Mmp2, Mmp9, Mmp28) showing mixed hormonal and genotype effects, but in aggregate less than 1.5 fold in any direction.  6.4 Conclusions We have assessed gene expression variability amongst Kiss1 and Gpr54 knockout mice to determine potential gene candidates involved in the regulation of this ligand-receptor pair. Initially an Affymetrix Exon 1.0 ST Array consisting of more than 1 million probe sets (exons) was used to assess gene expression differences between genotype. We re-validated the array estimated differences, using QPCR. This yielded a set of 70 gene loci with confirmed transcriptional differences between genotypes, which was supplemented with additional genes of interest and controls. However the differences observed could also be explained by secondary hormonal differences between wildtype and knockout  205 mice. To account for this hormonal feedback, all mice were castrated and treated with or without hormonal implant prior to gene expression comparisons between genotypes.  Considering the results of the hormone/genotype experiment, the observations of strong regulation for Gnrhr and Kiss1 show that the relevant region of the hypothalamus was sampled and that the methods used were capable of picking up transcriptional differences in two genes known to be strongly hormonally regulated. We have shown for the first time, counter regulation of Kiss1 in Gpr54 knockout mice. This is consistent with previous reports showing that upon interruption of the hypothalamic-pituitary-gonadal axis, Kiss1 is elevated in the hypothalamus of mice [21] and rats [30].  Relatively few large differences were seen in the initial Affymetrix array experiment, and fewer changes were seen in the Kiss1 knockout mice, overall, than in the Gpr54 knockout mice. Although both genotypes fail to undergo puberty, it has been reported that the kisspeptin loss of function alleles may be less penetrant than the Gpr54 knockout mice [31]. Endogenous basal activity of the GPR54 receptor could explain such a difference, as could the existence of an as yet unknown weaker binding peptide ligand for GPR54.  Some of the gene expression differences could be cell type specific. The hypothalamus is a relatively small region in the brain and is divided into discrete nuclei. These nuclei consist of a cluster of cells responsible for mediating various physiological functions. It has been established that the anteroventral periventricular nucleus (AVPV) and the arcuate nucleus (ARC) are the key  206 regions in the rodent hypothalamus involved in Kiss1 expression [16, 18-21, 32]. It is possible that the gene expression differences observed are neuron, specific reflecting only a few cells. Nevertheless, we were able to observe robust regulation of Gnrhr, which is predominantly restricted to the 500 GnRH expressing neurons in the hypothalamus, which suggests that the experiment was powered to detect transcription changes in at least this number of cell bodies. Small differences, although statistically significant, should be cautiously interpreted and considering the degree of variation seen with the load control estimates for the samples, we did not consider absolute effects of less than 1.5 fold to be interpretable, even if statistically significant. It should also be noted that especially where interaction effects are seen, differences in secondary sexual maturation of the hypothalamus at the time of castration could account for the effect. For example, regulation of GnRH receptor levels may show a “priming” effect in wildtype castrate mice from prior exposure to testosterone, compared with knockouts. The reversed direction of hormonal effect for Gnrhr between GKO and WT is consistent with such an explanation [33].  We did not compare directly the controlled group of castrate T+, T- mice with the intact knockout mice. Although the intact knockout mice are also sexually immature, the intact immature testis will still have active feedback to the hypothalamus and transcription differences would be expected. Indeed, a greater difference in Kiss1 expression was seen in the intact GKO mice than the castrate GKO mice. This is consistent with reports showing that Kiss1 expression is upregulated in the hypothalamus of castrated rodents [12, 21].  207 It has been shown that Kp can downregulate MMP9 expression [34]. We observe that Mmp9 is upregulated when there is Kiss1 loss in the KKO mice and the expected downregulation is observed when there is gain of Kiss1 expression, as we see in the GKO mice, however these observations are specific to the hormonally treated group for both mutants.  Similarly MMP2 has been shown to be downregulated by Kp [35] and in our study Mmp2 is downregulated in the highly expressing Kiss1 GKO mice, although at a fold change less than 1.5. Additionally, reciprocal up-regulation is absent in the KKO mice. Although it has been demonstrated that MMP9/2 can cleave and inactivate Kp, the precise relationship between the MMPs and Kp remains to be elucidated. As Mmp28 showed the greatest fold change difference in the GKO genotype only effect, with a fold change of 1.48, there may be a novel MMP involved in Kp-GPR54 regulation. Although all the MMPs continually appeared as significant transcription changes in our analysis, the fold change values were less than 1.5 for Mmp2 and Mmp28 and hence these results should be interpreted with caution. Another potentially Kp modifying gene is Klk1b22, which is downregulated greater than 2 fold in the hormone only effect in the GKO mice yet has one of the largest fold change in the GKO mice and the largest fold change in the KKO mice for the genotype and hormone interaction comparison. From the family of kallikreins, this translated peptide may have a role in processing the 145 aa KiSS1 protein to the active kisspeptins. The Klk1b22 sequence is one that would recognize and cleave an Arginine-Serine bond. This arrangement of amino acids  208 is found in the mouse sequence for the Kiss1 peptide; that requires cleavage to created the N-terminus of the 52 aa mouse equivalent of the human Kp-54. The precise Klk1b22 gene is not found in humans, however neither is the R-S bond in the KiSS1 peptide as a putative cleavage site. Rather, the bond that is cleaved in the human sequence is between R-G to create the N-terminus of Kp-54 [36, 37]. To address the possibility of Klk1b22 as a Kp modifying enzyme, one could incubate active Klk1b22 with full-length mouse Kiss1 peptide and perform mass spectrometry on the resulting products. Tmem144 was the only gene that was consistently upregulated in the KKO mice as compared with WT mice regardless of treatment. Tmem144 has the sequence of a 10-transmembrane receptor that can form ion transport and peptide channels. Non-selective cation channels and potassium channels have been shown to be necessary for Kp to depolarize GnRH neurons [38]. As Tmem144 is consistently upregulated in mice lacking Kiss1 expression, and GPR54 is co-localized to GnRH neurons [10], it may be that Tmem144 is the cation channel involved in GnRH neuronal activation through GPR54 stimulation. The transcription factor gene Npas4 was upregulated in the GKO mice with one of the largest fold changes after Kiss1, yet was downregulated by 1.65 fold in castrated mice independent of hormonal feedback. Npas4 has been credited with regulating the development of inhibitory synapses in activating neurons in the hippocampus [39]. What is most striking is that Npas4 is activated by an influx of calcium [39] and is independent of the MAP kinase pathway [40]. It has been recently reported that Kp-GPR54 signalling in the hypothalamus of rats  209 is independent of the MAP kinase pathway as well [38]. These results suggest that Npas4 may also have a role in Kp-GPR54 signalling, specifically as a transcription factor downstream of GPR54 activation that may induce GnRH release. However, as Npas4 is involved in neuronal maturation, the gene expression variability we are seeing between GKO and WT may be a result of the sexually immature phenotype of the knockout mice. Expression of the estrogen receptor beta (Esr2) was downregulated by 1.63 fold in the genotype only effect GKO mice. Similarly, the androgen receptor (Ar) was also hormonally independent in KKO mice, however with a fold change less than 1.2. Esr2 function in the hypothalamic-pituitary-gonadal axis is currently unknown as hormonal feedback occurs almost entirely through estrogen receptor alpha (Esr1), [16, 18, 21] as supported in our study. This feedback is also partially regulated through the Ar in male mice [21]. This is the first report that Esr2 may be involved in the hypothalamic-pituitary-gonadal axis, as regulated by Kp and GPR54. We compared Kiss1 and Gpr54 knockout mice hypothalamic gene expression to determine novel genes involved in the endocrine signalling pathway, however we were anticipating that novel genes also involved in the anti-metastatic pathway of Kp-GPR54 epithelial signalling may also arise. One such candidate is Wnt5a. In the GKO mice Wnt5a expression was decreased as compared with wildtype, although at levels lower than 1.5 fold, and this was independent of hormonal influence. Wnt5a signals in the non-canonical pathway and has been suggested to decrease Kp expression in melanoma cells thus  210 inducing the metastatic potential of these cells [41]. Furthermore, Wnt5a is an independent marker for poor prognosis in melanoma [42], the disease that first characterized Kp as a metastasis supressor [43]. Most recently, Wnt5a has been implicated in embryo implantation yet is not affected by hormones [44]. We propose that Wnt5a and the Kp-GPR54 ligand-receptor pair may play a role in trophoblast invasion in the placenta that is a fine balance between the regulation of these genes.  In our study we have determined a panel of genes that are involved in the regulation of the Kp-GPR54 ligand-receptor pair independent of, or in addition to, hormonal effects. However, further analysis is required to definitively include these candidates as part of either the endocrine or autocrine pathways regulated by Kiss1 and Gpr54.   211 6.5 References 1. de Roux N, Genin E, Carel JC, Matsuda F, Chaussain JL, Milgrom E: Hypogonadotropic hypogonadism due to loss of function of the KiSS1-derived peptide receptor GPR54. Proc Natl Acad Sci U S A 2003, 100(19):10972-10976. 2. Seminara SB, Messager S, Chatzidaki EE, Thresher RR, Acierno JS, Jr., Shagoury JK, Bo-Abbas Y, Kuohung W, Schwinof KM, Hendrick AG et al: The GPR54 gene as a regulator of puberty. N Engl J Med 2003, 349(17):1614-1627. 3. Arreguin-Arevalo JA, Lents CA, Farmerie TA, Nett TM, Clay CM: KiSS-1 peptide induces release of LH by a direct effect on the hypothalamus of ovariectomized ewes. In: Anim Reprod Sci. vol. 101; 2007: 265-275. 4. Castellano JM, Navarro VM, Fernandez-Fernandez R, Castano JP, Malagon MM, Aguilar E, Dieguez C, Magni P, Pinilla L, Tena-Sempere M: Ontogeny and mechanisms of action for the stimulatory effect of kisspeptin on gonadotropin-releasing hormone system of the rat. Mol Cell Endocrinol 2006, 257-258:75-83. 5. Dhillo WS, Chaudhri OB, Patterson M, Thompson EL, Murphy KG, Badman MK, McGowan BM, Amber V, Patel S, Ghatei MA et al: Kisspeptin-54 stimulates the hypothalamic-pituitary gonadal axis in human males. J Clin Endocrinol Metab 2005, 90(12):6609-6615. 6. Dhillo WS, Chaudhri OB, Thompson EL, Murphy KG, Patterson M, Ramachandran R, Nijher GK, Amber V, Kokkinos A, Donaldson M et al: Kisspeptin-54 stimulates gonadotropin release most potently during the preovulatory phase of the menstrual cycle in women. In: J Clin Endocrinol Metab. vol. 92; 2007: 3958-3966. 7. Gottsch ML, Cunningham MJ, Smith JT, Popa SM, Acohido BV, Crowley WF, Seminara SB, Clifton DK, Steiner RA: A role for kisspeptins in the regulation of gonadotropin secretion in the mouse. In: Endocrinology. vol. 145; 2004: 4073-4077. 8. Irwig MS, Fraley GS, Smith JT, Acohido BV, Popa SM, Cunningham MJ, Gottsch ML, Clifton DK, Steiner RA: Kisspeptin activation of gonadotropin releasing hormone neurons and regulation of KiSS-1 mRNA in the male rat. Neuroendocrinology 2004, 80(4):264-272. 9. Matsui H, Takatsu Y, Kumano S, Matsumoto H, Ohtaki T: Peripheral administration of metastin induces marked gonadotropin release and  212 ovulation in the rat. Biochem Biophys Res Commun 2004, 320(2):383- 388. 10. Messager S, Chatzidaki EE, Ma D, Hendrick AG, Zahn D, Dixon J, Thresher RR, Malinge I, Lomet D, Carlton MB et al: Kisspeptin directly stimulates gonadotropin-releasing hormone release via G protein- coupled receptor 54. Proc Natl Acad Sci U S A 2005, 102(5):1761-1766. 11. Navarro VM, Castellano JM, Fernández-Fernández R, Tovar S, Roa J, Mayen A, Barreiro ML, Casanueva FF, Aguilar E, Dieguez C et al: Effects of KiSS-1 peptide, the natural ligand of GPR54, on follicle-stimulating hormone secretion in the rat. In: Endocrinology. vol. 146; 2005: 1689- 1697. 12. Navarro VM, Castellano JM, Fernández-Fernández R, Tovar S, Roa J, Mayen A, Nogueiras R, Vazquez MJ, Barreiro ML, Magni P et al: Characterization of the potent luteinizing hormone-releasing activity of KiSS-1 peptide, the natural ligand of GPR54. In: Endocrinology. vol. 146; 2005: 156-163. 13. Plant TM: The role of KiSS-1 in the regulation of puberty in higher primates. Eur J Endocrinol 2006, 155 Suppl 1:S11-16. 14. Shahab M, Mastronardi C, Seminara SB, Crowley WF, Ojeda SR, Plant TM: Increased hypothalamic GPR54 signaling: a potential mechanism for initiation of puberty in primates. In: Proc Natl Acad Sci USA. vol. 102; 2005: 2129-2134. 15. Thompson EL, Murphy KG, Patterson M, Bewick GA, Stamp GW, Curtis AE, Cooke JH, Jethwa PH, Todd JF, Ghatei MA et al: Chronic subcutaneous administration of kisspeptin-54 causes testicular degeneration in adult male rats. In: Am J Physiol Endocrinol Metab. vol. 291; 2006: E1074-1082. 16. Adachi S, Yamada S, Takatsu Y, Matsui H, Kinoshita M, Takase K, Sugiura H, Ohtaki T, Matsumoto H, Uenoyama Y et al: Involvement of anteroventral periventricular metastin/kisspeptin neurons in estrogen positive feedback action on luteinizing hormone release in female rats. In: J Reprod Dev. vol. 53; 2007: 367-378. 17. Clarkson J, d'Anglemont de Tassigny X, Moreno AS, Colledge WH, Herbison AE: Kisspeptin-GPR54 signaling is essential for preovulatory gonadotropin-releasing hormone neuron activation and the luteinizing hormone surge. In: J Neurosci. vol. 28; 2008: 8691-8697. 18. Kauffman AS, Gottsch ML, Roa J, Byquist AC, Crown A, Clifton DK, Hoffman GE, Steiner RA, Tena-Sempere M: Sexual differentiation of  213 Kiss1 gene expression in the brain of the rat. In: Endocrinology. vol. 148; 2007: 1774-1783. 19. Smith JT, Cunningham MJ, Rissman EF, Clifton DK, Steiner RA: Regulation of Kiss1 gene expression in the brain of the female mouse. In: Endocrinology. vol. 146; 2005: 3686-3692. 20. Kinoshita M, Tsukamura H, Adachi S, Matsui H, Uenoyama Y, Iwata K, Yamada S, Inoue K, Ohtaki T, Matsumoto H et al: Involvement of central metastin in the regulation of preovulatory luteinizing hormone surge and estrous cyclicity in female rats. Endocrinology 2005, 146(10):4431- 4436. 21. Smith JT, Dungan HM, Stoll EA, Gottsch ML, Braun RE, Eacker SM, Clifton DK, Steiner RA: Differential regulation of KiSS-1 mRNA expression by sex steroids in the brain of the male mouse. In: Endocrinology. vol. 146; 2005: 2976-2984. 22. d'Anglemont de Tassigny X, Fagg LA, Dixon JP, Day K, Leitch HG, Hendrick AG, Zahn D, Franceschini I, Caraty A, Carlton MB et al: Hypogonadotropic hypogonadism in mice lacking a functional Kiss1 gene. Proc Natl Acad Sci U S A 2007, 104(25):10714-10719. 23. Ebling FJ, Brooks AN, Cronin AS, Ford H, Kerr JB: Estrogenic induction of spermatogenesis in the hypogonadal mouse. Endocrinology 2000, 141(8):2861-2869. 24. Wu Z, Irizarry, R.A., Gentleman, R., Murillo, F.M., and Spencer, F.: A Model Based Background Adjustment for Oligonucleotide Expression Arrays. Journal of the American Statistical Association 2004, 99(468):909–917. 25. Applied Biosystems: Relative Quantitation Using Comparative Ct: Getting Started Guide. In: 7900HT Fast Real-Time PCR System. Edited by Biosystems A. Foster City, California; 2005. 26. Chernoff H, Lehmann, EL: The use of maximum likelihood estimates in chi square tests for goodness-of-fit. Ann Math Stat 1954, 25:576-586. 27. Hochberg Y, Benjamini Y: More powerful procedures for multiple significance testing. Stat Med 1990, 9(7):811-818. 28. Linear mixed model implementation in lme4 [http://cran.r- project.org/web/packages/lme4/vignettes/Implementation.pdf] 29. R: A language and environment for statistical computing [http://www.R-project.org]  214 30. Navarro VM, Castellano JM, Fernández-Fernández R, Barreiro ML, Roa J, Sánchez-Criado JE, Aguilar E, Dieguez C, Pinilla L, Tena-Sempere M: Developmental and hormonally regulated messenger ribonucleic acid expression of KiSS-1 and its putative receptor, GPR54, in rat hypothalamus and potent luteinizing hormone-releasing activity of KiSS-1 peptide. In: Endocrinology. vol. 145; 2004: 4565-4574. 31. Lapatto R, Pallais JC, Zhang D, Chan YM, Mahan A, Cerrato F, Le WW, Hoffman GE, Seminara SB: Kiss1-/- mice exhibit more variable hypogonadism than Gpr54-/- mice. In: Endocrinology. vol. 148; 2007: 4927-4936. 32. Clarkson J, Herbison AE: Postnatal development of kisspeptin neurons in mouse hypothalamus; sexual dimorphism and projections to gonadotropin-releasing hormone neurons. In: Endocrinology. vol. 147; 2006: 5817-5825. 33. Negri-Cesi P, Colciago A, Pravettoni A, Casati L, Conti L, Celotti F: Sexual differentiation of the rodent hypothalamus: hormonal and environmental influences. J Steroid Biochem Mol Biol 2008, 109(3- 5):294-299. 34. Yan C, Wang H, Boyd DD: KiSS-1 represses 92-kDa type IV collagenase expression by down-regulating NF-kappa B binding to the promoter as a consequence of Ikappa Balpha -induced block of p65/p50 nuclear translocation. J Biol Chem 2001, 276(2):1164-1172. 35. Yoshioka K, Ohno Y, Horiguchi Y, Ozu C, Namiki K, Tachibana M: Effects of a KiSS-1 peptide, a metastasis suppressor gene, on the invasive ability of renal cell carcinoma cells through a modulation of a matrix metalloproteinase 2 expression. In: Life Sci. 2008. 36. Castano JP, Martinez-Fuentes AJ, Gutierrez-Pascual E, Vaudry H, Tena- Sempere M, Malagon MM: Intracellular signaling pathways activated by kisspeptins through GPR54: do multiple signals underlie function diversity? Peptides 2009, 30(1):10-15. 37. Nash KT, Welch DR: The KISS1 metastasis suppressor: mechanistic insights and clinical utility. Front Biosci 2006, 11:647-659. 38. Liu X, Lee K, Herbison AE: Kisspeptin excites gonadotropin-releasing hormone (GnRH) neurons through a phospholipase C / calcium- dependent pathway regulating multiple ion channels. In: Endocrinology. 2008. 39. Lin Y, Bloodgood BL, Hauser JL, Lapan AD, Koon AC, Kim TK, Hu LS, Malik AN, Greenberg ME: Activity-dependent regulation of inhibitory synapse development by Npas4. Nature 2008, 455(7217):1198-1204.  215 40. Coba MP, Valor LM, Kopanitsa MV, Afinowi NO, Grant SG: Kinase networks integrate profiles of N-methyl-D-aspartate receptor- mediated gene expression in hippocampus. J Biol Chem 2008, 283(49):34101-34107. 41. Dissanayake SK, Wade M, Johnson CE, O'Connell MP, Leotlela PD, French AD, Shah KV, Hewitt KJ, Rosenthal DT, Indig FE et al: The Wnt5A/protein kinase C pathway mediates motility in melanoma cells via the inhibition of metastasis suppressors and initiation of an epithelial to mesenchymal transition. J Biol Chem 2007, 282(23):17259-17271. 42. Da Forno PD, Pringle JH, Hutchinson P, Osborn J, Huang Q, Potter L, Hancox RA, Fletcher A, Saldanha GS: WNT5A expression increases during melanoma progression and correlates with outcome. Clin Cancer Res 2008, 14(18):5825-5832. 43. Lee JH, Miele ME, Hicks DJ, Phillips KK, Trent JM, Weissman BE, Welch DR: KiSS-1, a novel human malignant melanoma metastasis- suppressor gene. J Natl Cancer Inst 1996, 88(23):1731-1737. 44. Hayashi K, Erikson DW, Tilford SA, Bany BM, Maclean Ii JA, Rucker Iii EB, Johnson GA, Spencer TE: Wnt Genes in the Mouse Uterus: Potential Regulation of Implantation. Biol Reprod 2009.     216 7 SUMMARY, OVERALL CONCLUSIONS, AND FUTURE DIRECTIONS  7.1 Summary Kp and GPR54 have been studied as anti-metastatic agents [1-6] and have been determined to initiate sex hormone release through activation of the hypothalamic-pituitary-gonadal axis [7-15]. This dissertation hypothesized that expression of Kp and GPR54 would be predictive of disease outcome and anti- metastatic behaviour would be altered with the loss of GPR54 in a malignant epithelial cell line. An existing tumour that has Kp expression may inhibit metastasis through autocrine signalling assuming concurrent expression of GPR54 within the tumour. An overarching hypothesis would be that the secreted Kp may be released back into the system from this same tumour and stimulate sex hormone production through an endocrine pathway.  To address our hypothesis that Kp and GPR54 expression predict clinical outcome we used IHC on tissue microarrays. Prior to my research, Kp and GPR54 expression had been assessed in a number of cancers, usually using RNA in situ hybridization or QPCR technology, and that loss of either Kp and/or GPR54 was equated to a poorer outcome [16-21]. However, assessment of Kp and GPR54 expression by immunohistochemical methods had not been accomplished successfully. Although a few cases of breast and ovarian cancers have been studied, large cohorts with outcome data have not been used.  217 Furthermore, this ligand-receptor pair had not been studied in prostate or testicular cancers prior to this dissertation. We have established, using the antibodies and protocols optimized as describe in the thesis, the pattern of immunoreactivity for Kp and GPR54 in breast, ovarian, testicular, and prostate cancers. Specifically we have determined that: (1) loss of GPR54 is associated with poor prognosis in node positive breast cancer patients, (2) that seminomas express higher levels of GPR54 and Kp as compared with the more malignant testicular cancers, (3) that GPR54 is highly expressed in benign prostatic hyperplasia as compared to cancer, and (4) that both GPR54 and Kp are independent markers for favourable prognosis in ovarian cancer. Additionally we have correlated Kp and GPR54 expression with the clear cell subtype of ovarian cancer, further describing the least characterized ovarian cancer subtype. These favourable outcome associations are consistent with the literature and consistent with the notion that Kp and GPR54 may have a role in tumour migration, but perhaps not tumour initiation. Furthermore, we have shown for the first time, the range of tumours that may co-express GPR54 and Kp. An important caveat to these studies is the specificity of the antibodies used. Although clinical outcome data for the patients from the testicular germ cell array and the prostate array was unavailable, we can infer that GPR54 expression may correlate with a more favourable prognosis based on GPR54 expression specific to subtype. Seminomas have a more favourable outcome compared to non-seminomas and the seminomas in our array highly express GPR54 and Kp as compared to the more malignant testicular cancers. For Kp,  218 specificity was determined by examining the ability of blocking peptides to abrogate binding in the IHC pattern and also by examining pattern specific expression in placental tissue, where the expression of GPR54 and kisspeptin has been localized [2, 6, 22]. GPR54 specificity was determined by western blot, and revealed one clear band at 42 kD, the established protein weight of GPR54. These controls show that the antibodies and protocol used recognize GPR54 and Kp, but they do not completely exclude other proteins that may also be bound (at unknown affinity) by these antibodies. As Kp is a secreted peptide, we set out to determine whether Kp expression in tumours is then secreted into the bloodstream. Using plasma samples from a variety of gynaecological cancer patients, we were able to determine that Kp is indeed secreted into the bloodstream of these patients. Previously it had been determined that Kp is barely measurable in human plasma under normal physiological conditions except in pregnancy where it is dramatically elevated [23-27]. Two other groups also established plasma Kp elevation in cancer patients throughout the course of my research; in gestational trophoblastic neoplasia (GTN) [25] and in pancreatic cancer [28]. In GTN, elevated Kp returns to normal physiological levels after surgical removal of the tumour and chemotherapy. In pancreatic cancer patients, plasma Kp is elevated as compared to normal healthy volunteers, yet there is no correlation with any prognostic markers such as survival, grade or stage. Similarly, we also determined that Kp was elevated in plasma of gynaecological cancer patients but did not have the benefit of post-treatment blood samples to determine disease  219 specificity. However, there were significantly higher Kp levels in patients with a metastasis to the ovary than those that had a history of cancer, suggesting that plasma Kp does return to near physiological levels after treatment. Following that Kp is expressed in tumours and can be released into the bloodstream of cancer patients, we could hypothesize further whether Kp could then act upon the hypothalamus in an endocrine manner. Our group did not specifically assess whether plasma Kp could affect the hypothalamic-pituitary- gonadal axis through neuronal GPR54. Throughout the course of my research, other groups had determined that when peripheral Kp is injected into mammals, there is desensitization of the Kp-GPR54 endocrine axis in vivo [29, 30]. Furthermore, direct stimulation of GnRH neurons with Kp in vitro produced desensitization of these neurons for at least 20-30 min [31] thus we could hypothesize that plasma Kp when released from cancer would not be able to have a long term effect on hypothalamic signalling. However, if there is pulsatile Kp released from the tumour cell then it may be possible that there is still stimulation of the hypothalamic-pituitary-axis, as the desensitization of the GnRH neurons is temporary [29, 31]. Our second hypothesis was that anti-metastatic behaviour would be altered with the loss of GPR54 in a malignant epithelial cell line. Whether signalling desensitization occurs in malignant cells expressing GPR54 was/is unknown, however chronic expression of Kp in animal xenograft models of melanoma and breast cancer metastasis has been shown capable of reducing cell migration [3, 4].  My objective was to manipulate GPR54 signalling in breast  220 and/or ovarian cancer cell lines, to assess possible anti-metastatic effects and the role of autocrine signalling, if any. Several studies [1, 2, 5, 6, 32, 33] have examined the consequences of gain of function (forced overexpression) of GPR54 in metastatic cell lines and shown anti-migratory effects. What was not known, certainly for breast and ovarian cancers, is whether manipulation of endogenous GPR54 expression would result in altered migratory behaviour. To address this we were first required to survey breast and ovarian cancer cell lines for GPR54 expression and secondly, to show that downregulation of GPR54 could alter signalling. We were able to show expression of GPR54 in several breast and ovarian cancer cell lines, including exceptionally high expression in MCF-7 cells. The first important discovery was that none of these cell lines signal through the canonical calcium mobilization pathway, although some showed pERK1/2 responses. However when we attempted to manipulate GPR54 protein levels with shRNA constructs, it was discovered that in cell lines where strong knockdown of the protein could be observed, there was no significant alteration of MAPK signalling. While examining the effect of knockdown by immunofluorescence, it became apparent that GPR54 was not detectable on the plasma membrane. Rather, GPR54 was cytoplasmic in four of the endogenously expressing cell lines. GPR54 has been described as a G-protein coupled receptor that signals through the Gq calcium-releasing pathway [2, 5, 6]. However, upon Kp stimulation of the GPR54 endogenously expressing cells, there was no intracellular calcium release, consistent with the absence of FACS or IHC  221 detectable membrane expression. It remains possible that low levels of membranous GPR54 are present and the lack of calcium mobilization would be due to absence of the relevant intracellular G-proteins.  We were able to activate the MAP kinase pathway of the GPR54 internalized cells upon Kp stimulation, but for most of the cell lines, only by using higher than physiological levels of Kp. Whether there was true activation of the MAP kinase pathway via cytoplasmic GPR54 or whether very low levels of surface receptor are present, is unknown. We explored the possibility that the cytoplasmic receptor was the result of chronic endogenous activation by Kp, by knocking down Kiss1 transcripts by siRNA. Although GPR54 could be co-localized with -arrestin, the Kiss1 transcriptional knockdown did not result in re-localization of GPR54 to the membrane. It is important to consider that in Chapter 2 we describe that Kp IHC and Kiss1 RNA transcripts do not match. Therefore we acknowledge that the QPCR knockdown results of Kiss1 may inaccurately reflect Kp protein concentrations for this experiment. A full exploration of these observations would require the availability of a potent and specific small molecular inhibitor, however none have been described to date. Finally, it remains possible that the signalling seen through the MAPK pathway may represent activation (possible low affinity activation) of other peptide receptors, or the existence of an alternative receptor for kisspeptins, as has been speculated in the literature [34, 35]. Lastly, we set out to discover novel genes that may be involved in Kp and GPR54 signalling with the ultimate aim of comparing physiologically activated/repressed hypothalamic genes with those activated by GPR54  222 signalling in malignant cells. Since we could not show GPR54 specific signalling in the malignant cells, this comparison could not be made. Nevertheless, using genome wide transcript expression profiling methods we were able to show for the first time which gene loci have transcription that is dependent on intact Gpr54 and Kiss1 and which of these are likely hormonally regulated. Using Affymetrix Exon 1.0 Mouse ST Array hybridization of microdissected hypothalamic RNA, we first estimated differential gene expression between knockout and wildtype mice. Microdissection was used to limit the analysis to the anatomical region of the hypothalamus where GnRH and GPR54 expressing neurons exist. The initial analysis found 213 unique transcripts, to be differentially expressed between knockout and wildtype animals at greater than 1.5 fold. 70 of these genes were re-evaluated with an additional 25 controls and genes of interest that were not apparently differentially expressed on the Affymetrix arrays. This was achieved by using Low Density arrays; a 384 well probe-quenching QPCR platform. After this first pass analysis, 18 array derived gene loci survived re-evaluation by showing reproducible changes. These were carried forward with 25 additional control/interest genes to a final experiment where the effects of genotype and hormonal influence were separated. To achieve this, RNA from castrated male wildtype, Gpr54 knockout, and Kiss1 knockout mice that were implanted either with testosterone containing implants or vehicle only implants, was analyzed by QPCR. The results were analyzed using a 2x2x2 (genotype, hormone treatment, and test vs control groups) linear mixed effects model for the Gpr54 and Kiss1 knockouts.  223  From this analysis and addressing gene loci with greater than 1.5 fold change, we determined that from the Kiss1 knockout mice, only Gnrhr showed a pure hormonal effect and only Tmem144 showed a genotype effect. Among the genotype and hormone variant changes, Klk1b22 showed the greatest differences. For Gpr54 knockout mice, Klk1b22 showed a hormone only effect and a hormone effect with genotype interaction depending on the loading control. Gnrhr shows a strong hormonal effect with a genotype interaction while Tmem144 shows a strong genotype effect and a slight hormone effect without genotype interaction. Genotype only effects were seen with ER (Esr2), Hhip, Lhcgr, and Npas4. This is the first study to assess gene expression differences in Kiss1 and Gpr54 knockout mice and has created a list of gene candidates that should be further explored in their capacity to affect Kp-GPR54 signalling and regulation. We set out to identify novel gene candidates involved in Kp-GPR54 epithelial signalling using a neuronal system since the physiological role of Kp and GPR54 has been more definitively defined in the hypothalamus. Had we successfully created a GPR54 knockdown cell line model, we would have explored the gene candidates discovered in the neuronal system using the cell line model to determine if they are relevant in the Kp-GPR54 epithelial signalling.  7.2 Significance and Potential Applications The importance of considering both the neuroendocrine and epithelial characteristics of Kp and GPR54 arise when assessing Kp as a potential  224 therapeutic or diagnostic marker. Firstly, Kp could be considered as a potential therapeutic due to its anti-metastatic properties. It is known from previous studies that peripheral injections of Kp can stimulate the hypothalamic axis due to the increased levels of LH and FSH in humans after injection [23, 24], and furthermore there were no adverse affects in humans. As it has also been reported that GPR54 becomes desensitized to Kp after prolonged exposure [29- 31], there is little risk of increasing sex steroid levels by activating the hypothalamic-pituitary-gonadal axis through continuous administration of Kp. It is important to mention that there was testicular degeneration in rats with a single bolus of Kp administered subcutaneously [36] but this could be avoided while maintaining maximal gondaotropin release by adjusting the dose. Further studies need to be conducted before confirming that Kp has no adverse affects in humans. The anti-metastatic behaviour of Kp and GPR54 overexpressing cell lines has been well established [1-6, 37, 38]. However, whether there are anti- proliferative effects of Kp activation of GPR54 are still in question. That the MAP kinase pathway is activated upon Kp stimulation of GPR54 suggests that proliferation is not inhibited. Regardless of the contradictory results for anti- proliferative behaviour using in vitro studies, Kp expression in clinical samples is higher than in normal tissues yet Kp expression maintains a favourable prognostic marker phenotype in all cancers except hepatocellular cancer. These characteristics of Kp follow that of a tumourigenic agent that has anti-metastatic properties and should be taken into consideration when assessing Kp as a  225 potential therapeutic. Therefore, although Kp may not be tumourigenic through stimulation of sex steroid release, it may still be tumourigenic via an unknown mechanism. It may not be advantageous to use Kp as an anti-metastatic agent if there is the risk of Kp inducing cell proliferation. However a rigorous study conducted by Nash et al. [38] suggests that Kp induces dormancy in in vivo studies. By overexpressing Kp in a highly malignant melanoma cell line, it was determined that these cells could migrate to the lungs of injected mice, yet these cells failed to colonize. Additionally it was determined that this failure to colonize was specific to the cell’s ability to secrete Kp, indicating the possibility of GPR54 paracrine signalling mediating the anti-metastatic effects rather than autocrine signalling. Due to the slight possibility of Kp and GPR54 having a proliferative effect, and as yet unknown potential degenerative effects, it is not likely that Kp will soon be used as a therapeutic.  However the use of Kp as a diagnostic marker may be plausible. Further studies attempting to measure Kp in plasma samples of cancer patients would be required to achieve this aim. Assuming Kp plasma levels were representative of Kp expression within the tumour, the plasma levels could then be correlated with outcome. Kp would be an ideal candidate as a diagnostic marker in neoplastic disease where early stage is difficult to detect, such as ovarian cancer. Ovarian cancer is problematic in that it is an internal organ and patients are usually unaware of their disease in the early stages since there are no obvious symptoms [39]. We have determined that plasma Kp is elevated in patients with ovarian cancer and other gyneacological diseases. Unfortunately, we did not  226 have a proper control group of healthy volunteers and could not confirm within our study that normal physiological levels of Kp are <2 pmol/l as has been previously reported with this assay [23-25]. A Kp plasma screen would give a significant advantage to the patient by early detection and facilitate treatment of this disease, however further analysis is required to determine if plasma Kp can be distinguished between malignancy and benign disease.  The major issue encountered throughout this thesis has been reproducible immunohistochemical analysis with respect to the Kp antibody. The KiSS1 antibody from Santa Cruz that was used in these IHC experiments yielded robust results on the human placenta that was used as a positive control. The placenta was the only tissue that had intense and reproducible staining; regardless of whether the assay was performed manually or using the automated Ventana system. The IHC variability between cancer cohorts cannot be simply attributed to tissue type, although it is most likely that in the case of prostate cancer Kp is expressed at extremely low levels. Kp consistently correlated with the ovarian clear cell subtype in two independent cohorts, however these results were more striking in the original ovarian array. In the gynaecological array used as a validation cohort, the statistical analysis was also performed by grouping Kp IHC scores +1 and +2 together for the positive group. This grouping produced more positive clear cell cancer cases than when the +2 cases were considered as positive alone. However, the IHC score of +1 consisted of any Kp staining and included cases that had very weak signal. It is not realistic that an IHC biomarker  227 would be useful when the pathologist is expected to consider even weak staining as positive. Furthermore, that Kp can be a favourable prognostic marker in one cohort of clear cell cancer patients yet not in a separate cohort of clear cell cancer patients from a different institution is worrying. Details of the treatment of these cohorts are unavailable to assess as a possible reason for the inconsistent results. One would not realistically expect that difference of tissue treatment within the same country could account for the IHC results, and most likely reflects lack of reproducibility due to the antibody.  7.3 Overall Conclusions In summary, we have determined that Kp and GPR54 expression does predict clinical outcome; this expression correlates with favourable prognosis in ovarian cancer and GPR54 loss correlates with poor prognosis in node positive breast cancer patients. Furthermore, Kp can be secreted into the plasma of patients with gynealogical disease. Although we were unable to directly address our second hypothesis that GPR54 loss would alter Kp-GPR54 signalling, we have demonstrated that endogenous GPR54 is internalized in cancer cell lines. Moreover we have demonstrated that this cytoplasmic GPR54 does not signal through the canonical Gq signalling pathway upon Kp stimulation. Finally, we have discovered novel genes involved in the regulation of the Kp-GPR54 system.   228 7.4 Future Directions Due to the internalized GPR54 encountered in our cancer cell lines, we did not successfully establish a GPR54 knockdown model from a cancer cell line expressing a functioning plasma membrane receptor. A cell line that endogenously expresses GPR54 on the plasma membrane would be required in order for this to be accomplished, and as yet none have been reported. However, few studies have attempted to localize endogenous GPR54 in cancer cells, yet the literature supports that there are several lines with endogenous GPR54 that could be explored [37, 40-43]. Upon discovery of such a cell line, stably transfected knockdowns could be created using short hairpin RNA. With the creation of this model, the effects of endogenous GPR54 signalling could be studied and compared directly to the absence of GPR54 signalling. In the literature it has been established that upon Kp stimulation, cells expressing GPR54 have less invasive ability. Similar studies could be conducted such as: matrigel invasion assays, wound healing assay, and migration chamber assays. Cell proliferation assays of this system would be informative and would help support whether or not the arrest of proliferation in GPR54 overexpressing cell lines is due to the presence of GPR54. Although the above model system would help determine the mechanism involved in the anti-metastatic autocrine signalling effect of GPR54 by Kp, it would also be useful to explore cytoplasmic GPR54 further. As this is the first description of cytoplasmic GPR54 expressed endogenously, it would be helpful to know how frequently GPR54 is located in the cytoplasm. Identifying a cell line  229 with endogenously expressing GPR54 located on the plasma membrane as we have described above would help to elucidate this. This may be a common state for GPR54 in cancer cell lines, as we have determined that this is the case in all four of the cell lines studied. Similarly, it would be informative to discover the mechanism behind GPR54 endogenous internalization. We have co-localized GPR54 with -arrestin, but GPR54 may also be encapsulated in an endocytic vesicle. Further co-localization immunofluorescent studies would help determine the exact nature of GPR54 internalization using antibodies specific for proteins involved in endosomic vesicles such as clatherin [44]. Further verification of - arrestin's role in GPR54 internalization would also be valuable. Using short interfering RNAs to reduce -arrestin expression may be sufficient to bring GPR54 back to the plasma membrane. If this were true, then it would be advisable to determine if returning GPR54 to the cell membrane is sufficient to activate calcium release upon Kp stimulation. The Affymetrix analysis described in Chapter 6 comparing gene expression differences between knockout and wildtype mice was performed in order to discover new genes involved in Kp-GPR54 signalling. We successfully validated several genes through real-time PCR after controlling for sex hormone influence and have identified multiple gene candidates that deserve greater scrutiny. Of these candidates we are particularly interested in the 10- transmembrane protein, Tmem144, and the transcription factor Npas4. Npas4 was recently described to have a role in inducing inhibitory synapse development, and is activated by calcium ions [45]. This group also has an  230 antibody available for Npas4, so a starting point would be to determine if GPR54 and Npas4 are co-expressed within the same neurons in the hypothalamus. As the Gpr54 promoter is paired with an IRESlacZ sequence in the Gpr54 knockout mouse, there is -galactosidase activity in cells and tissues that would ordinarily express GPR54 [15]. The Npas4 antibody could be treated on hypothalamic tissues from the GPR54 knockout mouse to determine if there is co-localization with GPR54 expression. There are currently no publications describing Tmem144, however a similar study could be conducted as suggested for Npas4, as the Kiss1 knockout mouse was similarly created with a IRESlacZ sequence paired to the Kiss1 promoter [46]. In this instance one could develop a RNA in situ probe for Tmem144 that could be hybridized to hypothalamic tissues in the Kiss1 knockout mouse. However, in this case we would expect that Tmem144 would colocalize with Gpr54 expressing neurons if we hypothesize that cation released through a Tmem144 channel would stimulate GnRH neurons. Finally, a more comprehensive study assessing Kp in plasma of cancer patients would be extremely valuable. Such a study should be designed to include a true normal group of healthy volunteers as in our experiment all patients either had or were likely to have been affected by disease. Patients with benign disease should still be included, and all patients that are diagnosed with a malignancy. Furthermore, it would be extremely valuable to have a post- treatment sample from all patients regardless of their disease to establish whether Kp is disease specific. It has been established that plasma Kp returns to  231 normal after the delivery of the placenta in the case of pregnancy [25, 26] as well as after removal and treatment of the tumour in gestational trophoblast neoplasia [25]. Ours is the first report of plasma Kp elevation in benign disease, yet this may still be disease specific and once treated, plasma Kp may again return to normal physiological levels. Elevation of Kp in benign disease does not eliminate Kp as a potential plasma biomarker; rather it is still informative, as these benign diseases still require treatment. The reason for the elevated Kp levels in BRCA mutation carriers and those free of disease yet having a history of cancer is unexpected and potentially a cause for concern. It is the one resounding argument against using Kp as a potential plasma biomarker. However, these women should not be immediately disregarded and assumed free of disease. As it has been repeatedly reported that Kp at normal physiological levels is negligible [23-27], it is important to consider something more may be occurring with these patients. As the stress and risks associated with exploratory surgery and further testing in these patients may outweigh the potential benefits of discovering an unknown disease, it would be unethical at this point to consider contacting these women. However, it would be advisable to link any future reports of disease in these patients with their current results if possible. In summary, Kp and GPR54 are not currently diagnostic markers for malignancy, and though this ligand-receptor pair has the potential to become clinically useful, there is much more research required before this could be implemented.  232 7.5 References 1. Hori A, Honda S, Asada M, Ohtaki T, Oda K, Watanabe T, Shintani Y, Yamada T, Suenaga M, Kitada C et al: Metastin suppresses the motility and growth of CHO cells transfected with its receptor. Biochem Biophys Res Commun 2001, 286(5):958-963. 2. Kotani M, Detheux M, Vandenbogaerde A, Communi D, Vanderwinden JM, Le Poul E, Brézillon S, Tyldesley R, Suarez-Huerta N, Vandeput F et al: The metastasis suppressor gene KiSS-1 encodes kisspeptins, the natural ligands of the orphan G protein-coupled receptor GPR54. In: J Biol Chem. vol. 276; 2001: 34631-34636. 3. Lee JH, Miele ME, Hicks DJ, Phillips KK, Trent JM, Weissman BE, Welch DR: KiSS-1, a novel human malignant melanoma metastasis- suppressor gene. J Natl Cancer Inst 1996, 88(23):1731-1737. 4. Lee JH, Welch DR: Suppression of metastasis in human breast carcinoma MDA-MB-435 cells after transfection with the metastasis suppressor gene, KiSS-1. Cancer Res 1997, 57(12):2384-2387. 5. Muir AI, Chamberlain L, Elshourbagy NA, Michalovich D, Moore DJ, Calamari A, Szekeres PG, Sarau HM, Chambers JK, Murdock P et al: AXOR12, a novel human G protein-coupled receptor, activated by the peptide KiSS-1. J Biol Chem 2001, 276(31):28969-28975. 6. Ohtaki T, Shintani Y, Honda S, Matsumoto H, Hori A, Kanehashi K, Terao Y, Kumano S, Takatsu Y, Masuda Y et al: Metastasis suppressor gene KiSS-1 encodes peptide ligand of a G-protein-coupled receptor. Nature 2001, 411(6837):613-617. 7. Arreguin-Arevalo JA, Lents CA, Farmerie TA, Nett TM, Clay CM: KiSS-1 peptide induces release of LH by a direct effect on the hypothalamus of ovariectomized ewes. In: Anim Reprod Sci. vol. 101; 2007: 265-275. 8. Castellano JM, Navarro VM, Fernandez-Fernandez R, Castano JP, Malagon MM, Aguilar E, Dieguez C, Magni P, Pinilla L, Tena-Sempere M: Ontogeny and mechanisms of action for the stimulatory effect of kisspeptin on gonadotropin-releasing hormone system of the rat. Mol Cell Endocrinol 2006, 257-258:75-83. 9. Gottsch ML, Cunningham MJ, Smith JT, Popa SM, Acohido BV, Crowley WF, Seminara SB, Clifton DK, Steiner RA: A role for kisspeptins in the regulation of gonadotropin secretion in the mouse. In: Endocrinology. vol. 145; 2004: 4073-4077.  233 10. Matsui H, Takatsu Y, Kumano S, Matsumoto H, Ohtaki T: Peripheral administration of metastin induces marked gonadotropin release and ovulation in the rat. Biochem Biophys Res Commun 2004, 320(2):383- 388. 11. Messager S, Chatzidaki EE, Ma D, Hendrick AG, Zahn D, Dixon J, Thresher RR, Malinge I, Lomet D, Carlton MB et al: Kisspeptin directly stimulates gonadotropin-releasing hormone release via G protein- coupled receptor 54. Proc Natl Acad Sci U S A 2005, 102(5):1761-1766. 12. Navarro VM, Castellano JM, Fernández-Fernández R, Tovar S, Roa J, Mayen A, Barreiro ML, Casanueva FF, Aguilar E, Dieguez C et al: Effects of KiSS-1 peptide, the natural ligand of GPR54, on follicle-stimulating hormone secretion in the rat. In: Endocrinology. vol. 146; 2005: 1689- 1697. 13. Navarro VM, Castellano JM, Fernández-Fernández R, Tovar S, Roa J, Mayen A, Nogueiras R, Vazquez MJ, Barreiro ML, Magni P et al: Characterization of the potent luteinizing hormone-releasing activity of KiSS-1 peptide, the natural ligand of GPR54. In: Endocrinology. vol. 146; 2005: 156-163. 14. Plant TM: The role of KiSS-1 in the regulation of puberty in higher primates. Eur J Endocrinol 2006, 155 Suppl 1:S11-16. 15. Seminara SB, Messager S, Chatzidaki EE, Thresher RR, Acierno JS, Jr., Shagoury JK, Bo-Abbas Y, Kuohung W, Schwinof KM, Hendrick AG et al: The GPR54 gene as a regulator of puberty. N Engl J Med 2003, 349(17):1614-1627. 16. Dhar DK, Naora H, Kubota H, Maruyama R, Yoshimura H, Tonomoto Y, Tachibana M, Ono T, Otani H, Nagasue N: Downregulation of KiSS-1 expression is responsible for tumor invasion and worse prognosis in gastric carcinoma. Int J Cancer 2004, 111(6):868-872. 17. Ikeguchi M, Yamaguchi K, Kaibara N: Clinical significance of the loss of KiSS-1 and orphan G-protein-coupled receptor (hOT7T175) gene expression in esophageal squamous cell carcinoma. Clin Cancer Res 2004, 10(4):1379-1383. 18. Ringel MD, Hardy E, Bernet VJ, Burch HB, Schuppert F, Burman KD, Saji M: Metastin receptor is overexpressed in papillary thyroid cancer and activates MAP kinase in thyroid cancer cells. J Clin Endocrinol Metab 2002, 87(5):2399. 19. Sanchez-Carbayo M, Capodieci P, Cordon-Cardo C: Tumor suppressor role of KiSS-1 in bladder cancer: loss of KiSS-1 expression is  234 associated with bladder cancer progression and clinical outcome. Am J Pathol 2003, 162(2):609-617. 20. Shirasaki F, Takata M, Hatta N, Takehara K: Loss of expression of the metastasis suppressor gene KiSS1 during melanoma progression and its association with LOH of chromosome 6q16.3-q23. Cancer Res 2001, 61(20):7422-7425. 21. Stark AM, Tongers K, Maass N, Mehdorn HM, Held-Feindt J: Reduced metastasis-suppressor gene mRNA-expression in breast cancer brain metastases. J Cancer Res Clin Oncol 2005, 131(3):191-198. 22. Bilban M, Ghaffari-Tabrizi N, Hintermann E, Bauer S, Molzer S, Zoratti C, Malli R, Sharabi A, Hiden U, Graier W et al: Kisspeptin-10, a KiSS- 1/metastin-derived decapeptide, is a physiological invasion inhibitor of primary human trophoblasts. J Cell Sci 2004, 117(Pt 8):1319-1328. 23. Dhillo WS, Chaudhri OB, Patterson M, Thompson EL, Murphy KG, Badman MK, McGowan BM, Amber V, Patel S, Ghatei MA et al: Kisspeptin-54 stimulates the hypothalamic-pituitary gonadal axis in human males. J Clin Endocrinol Metab 2005, 90(12):6609-6615. 24. Dhillo WS, Chaudhri OB, Thompson EL, Murphy KG, Patterson M, Ramachandran R, Nijher GK, Amber V, Kokkinos A, Donaldson M et al: Kisspeptin-54 stimulates gonadotropin release most potently during the preovulatory phase of the menstrual cycle in women. In: J Clin Endocrinol Metab. vol. 92; 2007: 3958-3966. 25. Dhillo WS, Savage P, Murphy KG, Chaudhri OB, Patterson M, Nijher GM, Foggo VM, Dancey GS, Mitchell H, Seckl MJ et al: Plasma kisspeptin is raised in patients with gestational trophoblastic neoplasia and falls during treatment. Am J Physiol Endocrinol Metab 2006, 291(5):E878- 884. 26. Horikoshi Y, Matsumoto H, Takatsu Y, Ohtaki T, Kitada C, Usuki S, Fujino M: Dramatic elevation of plasma metastin concentrations in human pregnancy: metastin as a novel placenta-derived hormone in humans. J Clin Endocrinol Metab 2003, 88(2):914-919. 27. Katagiri F, Tomita K, Oishi S, Takeyama M, Fujii N: Establishment and clinical application of enzyme immunoassays for determination of luteinizing hormone releasing hormone and metastin. In: J Pept Sci. vol. 13; 2007: 422-429. 28. Katagiri F, Nagai K, Kida A, Tomita K, Oishi S, Takeyama M, Doi R, Fujii N: Clinical significance of plasma metastin level in pancreatic cancer patients. Oncol Rep 2009, 21(3):815-819.  235 29. Plant TM, Ramaswamy S, Dipietro MJ: Repetitive activation of hypothalamic G protein-coupled receptor 54 with intravenous pulses of kisspeptin in the juvenile monkey (Macaca mulatta) elicits a sustained train of gonadotropin-releasing hormone discharges. Endocrinology 2006, 147(2):1007-1013. 30. Thompson EL, Murphy KG, Patterson M, Bewick GA, Stamp GW, Curtis AE, Cooke JH, Jethwa PH, Todd JF, Ghatei MA et al: Chronic subcutaneous administration of kisspeptin-54 causes testicular degeneration in adult male rats. In: Am J Physiol Endocrinol Metab. vol. 291; 2006: E1074-1082. 31. Liu X, Lee K, Herbison AE: Kisspeptin excites gonadotropin-releasing hormone (GnRH) neurons through a phospholipase C / calcium- dependent pathway regulating multiple ion channels. In: Endocrinology. 2008. 32. Becker JA, Mirjolet JF, Bernard J, Burgeon E, Simons MJ, Vassart G, Parmentier M, Libert F: Activation of GPR54 promotes cell cycle arrest and apoptosis of human tumor cells through a specific transcriptional program not shared by other Gq-coupled receptors. In: Biochem Biophys Res Commun. vol. 326; 2005: 677-686. 33. Stathatos N, Bourdeau I, Espinosa AV, Saji M, Vasko VV, Burman KD, Stratakis CA, Ringel MD: KiSS-1/G protein-coupled receptor 54 metastasis suppressor pathway increases myocyte-enriched calcineurin interacting protein 1 expression and chronically inhibits calcineurin activity. In: J Clin Endocrinol Metab. vol. 90; 2005: 5432- 5440. 34. Harms JF, Welch DR, Miele ME: KISS1 metastasis suppression and emergent pathways. Clin Exp Metastasis 2003, 20(1):11-18. 35. Nash KT, Welch DR: The KISS1 metastasis suppressor: mechanistic insights and clinical utility. Front Biosci 2006, 11:647-659. 36. Thompson EL, Amber V, Stamp GW, Patterson M, Curtis AE, Cooke JH, Appleby GF, Dhillo WS, Ghatei MA, Bloom SR et al: Kisspeptin-54 at high doses acutely induces testicular degeneration in adult male rats via central mechanisms. Br J Pharmacol 2009, 156(4):609-625. 37. Masui T, Doi R, Mori T, Toyoda E, Koizumi M, Kami K, Ito D, Peiper SC, Broach JR, Oishi S et al: Metastin and its variant forms suppress migration of pancreatic cancer cells. Biochem Biophys Res Commun 2004, 315(1):85-92. 38. Nash KT, Phadke PA, Navenot JM, Hurst DR, Accavitti-Loper MA, Sztul E, Vaidya KS, Frost AR, Kappes JC, Peiper SC et al: Requirement of KISS1  236 secretion for multiple organ metastasis suppression and maintenance of tumor dormancy. J Natl Cancer Inst 2007, 99(4):309- 321. 39. Cannistra SA: Cancer of the ovary. N Engl J Med 2004, 351(24):2519- 2529. 40. Jiang Y, Berk M, Singh LS, Tan H, Yin L, Powell CT, Xu Y: KiSS1 suppresses metastasis in human ovarian cancer via inhibition of protein kinase C alpha. Clin Exp Metastasis 2005, 22(5):369-376. 41. Shoji S, Tang XY, Umemura S, Itoh J, Takekoshi S, Shima M, Usui Y, Nagata Y, Uchida T, Osamura RY et al: Metastin Inhibits Migration and Invasion of Renal Cell Carcinoma with Overexpression of Metastin Receptor. In: Eur Urol. 2008. 42. Yan C, Wang H, Boyd DD: KiSS-1 represses 92-kDa type IV collagenase expression by down-regulating NF-kappa B binding to the promoter as a consequence of Ikappa Balpha -induced block of p65/p50 nuclear translocation. J Biol Chem 2001, 276(2):1164-1172. 43. Yoshioka K, Ohno Y, Horiguchi Y, Ozu C, Namiki K, Tachibana M: Effects of a KiSS-1 peptide, a metastasis suppressor gene, on the invasive ability of renal cell carcinoma cells through a modulation of a matrix metalloproteinase 2 expression. In: Life Sci. 2008. 44. Moore CA, Milano SK, Benovic JL: Regulation of receptor trafficking by GRKs and arrestins. Annu Rev Physiol 2007, 69:451-482. 45. Lin Y, Bloodgood BL, Hauser JL, Lapan AD, Koon AC, Kim TK, Hu LS, Malik AN, Greenberg ME: Activity-dependent regulation of inhibitory synapse development by Npas4. Nature 2008, 455(7217):1198-1204. 46. d'Anglemont de Tassigny X, Fagg LA, Dixon JP, Day K, Leitch HG, Hendrick AG, Zahn D, Franceschini I, Caraty A, Carlton MB et al: Hypogonadotropic hypogonadism in mice lacking a functional Kiss1 gene. Proc Natl Acad Sci U S A 2007, 104(25):10714-10719.   

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