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Characterisation of the Direct Antiproliferative Effects of a Gonadotrophin-releasing Hormone Analogue Colette Meyer A thesis submitted in fulfilment of the requirements of the degree of Doctor of Philosophy The University of Edinburgh 2012 1 Abstract Gonadotrophin-releasing hormone (GnRH) can inhibit proliferation of multiple reproductive tissue cancer cell lines through direct interaction with GnRH receptors (GnRHR) on tumour cells. GnRH analogues may therefore have a role in treating some cancers. The signalling pathways associated with these inhibitory effects are poorly defined, and characterising them may help to understand therapeutic sensitivity. To elucidate these pathways, transcriptomic and proteomic approaches were used to compare the effects of the GnRH agonist Triptorelin in responsive GnRHR-transfected HEK293 cells (SCL60) and unresponsive (HEK293) cells both in vitro for up to 24h and in vivo for up to 7 days. Gene expression profiling demonstrated that SCL60 gene expression was temporally regulated with Triptorelin treatment, with expression of some genes increased at one time point but decreased at another. Early and mid-phase gene expression changes comprised mainly transcription factors and late changes included the hormonal signalling component CGA . Pathway analysis implicated mitogen-activated protein kinase and cell cycle pathways, supporting the detection of G 2/M arrest. Signalling effects within SCL60 xenografts, 4 and 7 days following Triptorelin treatment, were investigated using a phosphoproteomic antibody array. Changes included cell cycle and apoptosis regulators, as well as cell surface receptors and NF κB signalling pathway members. Reverse-phase protein arrays and western blotting also showed that pAkt was decreased and pNFκB-p65 was increased after Triptorelin treatment in vitro . An NF κB inhibitor enhanced the anti-proliferative effect of Triptorelin in SCL60 cells in vitro, suggesting that NF κB acts as a survival factor in the response to GnRHR stimulation. A range of GnRHR expression was observed in breast cancer tumours by immunohistochemistry, and on average GnRHR expression was significantly higher in the Triple Negative Phenotype (TNP) subgroup and in grade 3 tumours. A GnRHR-transfected breast cancer cell line, MCF7-h14, was developed. Despite this expressing a similar level of GnRHR to responsive SCL60 cells, MCF7-h14 cells were not inhibited by GnRHR activation, indicating that a high level of GnRHR is insufficient for the antiproliferative effects of Triptorelin. 2 Declaration The work in this thesis is solely my own work unless otherwise indicated, where credit to the contributor is given. No part of the work has been submitted for any other degree. Colette Meyer Acknowledgements I thank my supervisors Simon Langdon, Andy Sims, Kevin Morgan, Dana Faratian and David Harrison. I thank all my colleagues in the Breakthrough Research Unit, Edinburgh who have assisted me with experiments. I would also like to thank Kevin Morgan and Nicola Miller of the MRC Human Reproductive Sciences Unit, Queen’s Medical Research Institute, Edinburgh for their support and expertise. The SCL60 and SCL215 cell lines and their parental HEK293 cells were provided by Kevin Morgan. Mouse SCL60 and HEK293 xenograft experiments were performed by Morwenna Muir (University of Edinburgh Cancer Research Centre, Western General Hospital, Edinburgh) as a scientific service. Tissue from the SCL60 xenografts was used in immunohistochemical assays. Inhwa Um (Research Assistant, Breakthrough Research Unit, Edinburgh) constructed tissue microarrays from the SCL60 xenografts. Immunohistochemical staining for all markers except GnRHR was performed by myself, with the supervision of Simon Langdon, who also scored some of the slides. I prepared samples for flow cytometry analysis, which were processed on the flow cytometer by Elisabeth Freyer (Flow Cytometry Service, MRC Human Genetics Unit, Western General Hospital, Edinburgh) as a scientific service. 3 I designed the gene expression microarray study with the supervision of Simon Langdon and Andy Sims. I performed the cell treatments, RNA extractions, purifications and labelling. Alexey Larionov (Research Fellow, Breakthrough Research Unit, Edinburgh) provided advice on the labelling procedure. Charlene Kay (Research Assistant, Breakthrough Research Unit, Edinburgh), Annelein Zweemer (Student, Breakthrough Research Unit, Edinburgh) and Huizhong Hu (Student, Breakthrough Research Unit, Edinburgh) assisted with the procedure for biotin- labelling RNA under my direction. The microarrays were processed by the Wellcome Trust Clinical Research Facility, Western General Hospital, Edinburgh as a scientific service. I filtered and normalised the resultant data, and performed quality control checks. I performed the analyses to detect differential gene expression. SCL60 xenografts antibody arrays were performed by Eurogentec as a scientific service. They provided data for phosphorylated protein normalised to non- phosphorylated protein, which I analysed for significant differences between treatment groups. Peter Mullen (Research Assistant, Breakthrough Research Unit, Edinburgh) provided advice on processing the reverse-phase protein arrays. I designed the experiments, collected the protein, standardised the protein concentration across samples, performed the arrays and analysed the data. I performed all western blots in chapter 3.3. Ilgin Cagnan, a student working under my supervision, measured GnRHR expression by quantitative immunofluorescence. The tissue microarrays used were provided by Dana Faratian, and are detailed in the publication “Quantitative analysis of changes in ER, PR and HER2 expression in primary breast cancer and paired nodal metastases, Aitken et al , Ann Oncol, 2009” [1]. The resulting data was analysed by myself. Andy Sims and I both analysed the mRNA level of GnRHR in published datasets of breast cancers. Andy Sims provided Figure 61. 4 Kevin Morgan performed the stable transfection of breast cancer cell lines, and experiments in chapter 3.6 of this thesis were done collaboratively between Kevin Morgan and myself, with the exception of Figure 78 and Figure 84, which were provided by Kevin Morgan. Funding for this research project was provided by Breakthrough Breast Cancer. Part of this thesis is published in the BMC cancer paper: “GnRH receptor activation competes at a low level with growth signaling in stably transfected human breast cell lines, Morgan K, Meyer C, Miller N, Sims AH, Cagnan I, Faratian D, Harrison DJ, Millar RP, Langdon SP, 2011”. Kevin Morgan prepared the manuscript. Kevin Morgan, myself and Nicola Miller performed ligand binding assays, SRB assays and western blots. Ilgin Cagnan measured GnRHR primary breast tissue microarrays by immunofluorescence. I analysed the immunofluorescence data. Dana Faratian and provided tissue and clinical data for immunofluorescence analysis. Andy Sims, Dana Faratian, David Harrison, Robert Millar and Simon Langdon contributed intellectually, in supervision, and in manuscript preparation. 5 List of Contents Abstract ........................................................................................................................ 2 Declaration ................................................................................................................... 3 Acknowledgements...................................................................................................... 3 List of Contents............................................................................................................ 6 List of Figures ............................................................................................................ 11 List of Tables.............................................................................................................. 15 List of Abbreviations.................................................................................................. 16 1 Introduction............................................................................................................. 20 1.1 Breast cancer .................................................................................................... 20 1.1.1 The Hallmarks of Cancer .......................................................................... 20 1.1.2 Breast Biology........................................................................................... 22 1.1.3 Breast Cancer Background........................................................................ 23 1.1.3.1 Epidemiology....................................................................................