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This Electronic Thesis Or Dissertation Has Been Downloaded from Explore Bristol Research This electronic thesis or dissertation has been downloaded from Explore Bristol Research, http://research-information.bristol.ac.uk Author: Marcolino De Assis Junior, Eudmar Title: The regulation of PRH/HHEX by transforming growth factor General rights Access to the thesis is subject to the Creative Commons Attribution - NonCommercial-No Derivatives 4.0 International Public License. A copy of this may be found at https://creativecommons.org/licenses/by-nc-nd/4.0/legalcode This license sets out your rights and the restrictions that apply to your access to the thesis so it is important you read this before proceeding. Take down policy Some pages of this thesis may have been removed for copyright restrictions prior to having it been deposited in Explore Bristol Research. However, if you have discovered material within the thesis that you consider to be unlawful e.g. breaches of copyright (either yours or that of a third party) or any other law, including but not limited to those relating to patent, trademark, confidentiality, data protection, obscenity, defamation, libel, then please contact [email protected] and include the following information in your message: •Your contact details •Bibliographic details for the item, including a URL •An outline nature of the complaint Your claim will be investigated and, where appropriate, the item in question will be removed from public view as soon as possible. The regulation of PRH/HHEX by transforming growth factor β By Eudmar Marcolino de Assis Junior A thesis submitted to the University of Bristol, School of Biochemistry, for the degree of Doctor of Philosophy Word count: 56,442 ABSTRACT The transcription factor PRH/HHEX (Proline-Rich Homeodomain/Haematopoietically Expressed Homeobox) controls cell proliferation, cell differentiation and cell migration/invasion in a diverse range of cell types. Here I show that the gene encoding PRH is deleted in around 4% of sequenced samples from patients with prostate cancer. Additionally, I show that there is increased CpG DNA methylation at this gene in prostate cancer samples compared to samples from normal prostate and that PRH mRNA levels are reduced in prostate cancer. Using immunohistochemistry to stain 96 samples of prostate neoplasia, I show that PRH expression is negatively correlated to tumour differentiation in prostate cancer. Recent published work has shown that the down-regulation of PRH expression in breast and prostate cancer cells results in increased cell proliferation and increased cancer cell invasion. Moreover, PRH inhibits breast and prostate cancer cell migration and invasion in part at least by activating the transcription of Endoglin, a Transforming Growth Factor β (TGF-β) co-receptor. Here I show that the treatment of immortalised prostate epithelial (PNT2-C2) cells and prostate adenocarcinoma (PC3) cells with TGF-β increases cell migration and induces an epithelial to mesenchymal transition (EMT), with up-regulation of the EMT transcription factor Snail and down-regulation of the epithelial marker E-Cadherin. TGF-β treatment down-regulates the expression of PRH/HHEX in prostate cell lines at both the protein and mRNA level. Moreover, chromatin immunoprecipitation (ChIP) experiments show that the regulation of PRH/HHEX by TGF-β may be via the direct binding of the TGF-β effector pSmad3 at the PRH promoter. I also investigate the effects of PRH over-expression on PC3 cells. PRH expression in these cells re-activates the expression of E-cadherin, reduces the migratory ability of these cells and inhibits cell proliferation. PRH over-expression also down-regulates the expression of multiple genes involved in the TGF-β signalling pathway including TGFB2 and TGFBR2. ChIP shows that PRH binds to both the TGFB2 and TGFBR2 gene bodies, suggesting a direct effect of PRH on the expression of these genes. Prostate cancer cells are exposed to TGF-β from multiple sources in vivo and blood platelets were tested as a possible source of TGF- β using this model. Platelet treatment was able to increase cell migration, trigger EMT and down-regulate PRH expression in a similar way to TGF-β treatment. Platelets treatment resulted in the up-regulation of pSmad3 and the effect of platelets on cell migration were abolished when TGF-β signalling was blocked. In summary, the work described in this Thesis shows that TGF-β increases prostate cell migration in part at least by the down-regulation of PRH expression. PRH up- regulates E-cadherin expression and down-regulates cell migration suggesting that TGF-β alters E-cadherin levels and increases cell migration via its effects on PRH. Since PRH also regulates several genes important in TGF-β signalling this creates a feedback loop that enables a more precise control of cell behaviour. Changes in PRH levels or activity are likely to disrupt this control mechanism and to contribute to tumour progression. Acknowledgements I would like to say thank you, To mother, Sandra, for the unconditional love and support, without her I would not be here. To my family, especially my father and my siblings, Emanuel and Cinthya for the love and patience, I would not have the strength to carry on without them. To my supervisor, Prof. Kevin Gaston, for the guidance, patience, support and wisdom for these past four years. I learned so much from you I cannot thank you enough. To Dr. Sheela Jayaraman and her group, especially Dr. Philip Kitchen, who welcomed me into their group to learn new techniques that were essential to my thesis. To the D40 laboratory, you were an amazing support network, and were always there when I needed. I learned a lot from you. To my friends, Gabriel, Grace, Caitriona, Emma, Fruzsina, Lucy, Gemma, Jack, Alex, Anisha, Adriene, Jenifer, Gabriella, Emmanuelle, David and Andrew. You were like my family away from home. You love me and helped me when thing got very difficult. I truly love you all. Specially to Andrew and Adriene that helped me edit this thesis, I do not think I could had done without your help. To my friend in Brazil, Bruno, Gleyciane, Camila, Pedro, Debora, Evelin, Thayanne and my lovely god son Guilherme, who supported me coming to the UK, and who are there missing me and wishing great things for my future. I feel you close to my heart. To my previous supervisor, Prof. Roberto Cesar, thanks for introducing me to science and being a mentor to me. To the Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) through the Sciences Without Borders program, for the financial support for the project and for the scholarship. This is dedicated to my mother, whom is my everything. "It is our choices that show what we truly are, far more than our abilities." Albus Dumbledore AUTHOR’S DECLARATION I declare that the work in this dissertation was carried out in accordance with the requirements of the University‘s regulations and Code of Practice for Research Degree Programmes and that it has not been submitted for any other academic award. Except where indicated by specific reference in the text, the work is the candidate’s own work. Work done in collaboration with, or with the assistance of, others, is indicated as such. Any views expressed in the dissertation are those of the author. SIGNED:…………………………….DATE:……………………… Table of contents Chapter 1: Introduction ....................................................................................... 18 Cancer .................................................................................................................. 19 Hallmarks of cancer .......................................................................................... 20 Epithelial to mesenchymal transition..................................................................... 31 Epithelial tissue ................................................................................................. 31 EMT .................................................................................................................. 35 The prostate gland ................................................................................................ 44 Morphology and physiology .............................................................................. 44 Prostate cancer ................................................................................................. 46 Transforming growth factor β ................................................................................ 56 Platelets ................................................................................................................ 60 Proline Rich Homeodomain .................................................................................. 62 Aims ...................................................................................................................... 66 Chapter 2: Methodology ...................................................................................... 67 List of chemicals ................................................................................................... 68 Buffers .................................................................................................................. 71 Plasmids, primers and antibodies ......................................................................... 73 Experimental protocols ......................................................................................... 77 Plasmid amplification ........................................................................................ 77 Lentivirus production ........................................................................................
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