Targeting immediate early genes in melanoma and vascular diseases Ahmad Mohammad Nabil Alhendi A thesis submitted in fulfilment of the requirements for the degree of Doctorate of Philosophy (Biochemistry and Molecular Genetics) School of Biotechnology and Biomolecular Sciences The University of New South Wales March 2015 i ii ORIGINALITY STATEMENT ‘I hereby declare that this submission is my own work and to the best of my knowledge it contains no materials previously published or written by another person, or substantial proportions of material which have been accepted for the award of any other degree or diploma at UNSW or any other educational institution, except where due acknowledgement is made in the thesis. Any contribution made to the research by others, with whom I have worked at UNSW or elsewhere, is explicitly acknowledged in the thesis. I also declare that the intellectual content of this thesis is the product of my own work, except to the extent that assistance from others in the project's design and conception or in style, presentation and linguistic expression is acknowledged.’ Signed ……………………………… Date ………………………………….... iii COPYRIGHT STATEMENT ‘I hereby grant the University of New South Wales or its agents the right to archive and to make available my thesis or dissertation in whole or part in the University libraries in all forms of media, now or here after known, subject to the provisions of the Copyright Act 1968. I retain all proprietary rights, such as patent rights. I also retain the right to use in future works (such as articles or books) all or part of this thesis or dissertation. I also authorise University Microfilms to use the 350 word abstract of my thesis in Dissertation Abstract International (this is applicable to doctoral theses only). I have either used no substantial portions of copyright material in my thesis or I have obtained permission to use copyright material; where permission has not been granted I have applied/will apply for a partial restriction of the digital copy of my thesis or dissertation.' Signed ……………………………… Date ………………………………….... AUTHENTICITY STATEMENT ‘I certify that the Library deposit digital copy is a direct equivalent of the final officially approved version of my thesis. No emendation of content has occurred and if there are any minor variations in formatting, they are the result of the conversion to digital format.’ Signed ……………………………… Date ………………………………….... iv ABSTRACT Melanoma and cardiovascular disease (CVD) are highly prevalent diseases in Western nations. Disease initiation and progression occur via a number of different external and internal factors, often resulting in altered gene expression. The fastest response to such factors is the altered expression of the so-called immediate early genes (IEGs) and these genes are implicated in certain diseases including CVD and cancer. Dysregulation of IEGs in human aortic smooth muscle cells (hASMCs) usually results in a phenotypic transformation, involving the secretion of high levels of extracellular matrix proteins and increased proliferation and migration. In this study, 347 IEGs that were stress induced by addition of growth factor FGF2 and cytokine IL-1B and expressed in hASMCs, were categorised into three groups, early, rapid transient and late responder genes. Early growth response-1 (EGR1) an early gene and modulator of rapid transient genes NAB2, VCAM-1, ICAM-1 and late responder genes C-JUN and JUND was knocked down and the effect (down-regulation) on other IEG groups evaluated. Two miRNAs of interest with potential roles in cellular process relevant to melanoma are miR-155 and miR-125b. We demonstrated that the stress inducible transcription factors, C- JUN and C-FOS in melanoma cells are regulated by miR-125b and miR-155. Melanoma cell proliferation, migration and cell cycle analysis were performed to validate the effect of the two miRNAs on C-JUN and C-FOS expression. miRNAs caused decrease in proliferation, migration and affected cell cycle progression. The effect of miRNAs on C-JUN and C-FOS is promising in terms of down-regulating these transcription factors, which are usually upregulated in cancers. In parallel, the lead compound termed X compound, and its derivatives X4, X6, X7, BT2 and BT3, were tested on a melanoma cell line to assess their potential as chemotherapeutic agents. Treatment with these compounds resulted in cancer relevant phenotypic changes such as decreased expression of genes that are associated with melanoma, which suggest that the X compound, and its derivatives, should be explored as therapeutic agents to treat melanoma. Melanoma and CVD are so prevalent, thus, finding additional therapeutic alternatives is of great importance. v ACKNOWLEDGEMENTS First and foremost, I would like to express the deepest appreciation to my supervisor Noel for his guidance and mentorship for the past 2 years. Also, I would like to thank my co- supervisor Lionel for your assistance. Thanks for answering all those questions I had. Thank you both for always looking out for me and keeping my best interests at heart. I also would like to thank Levon for the great opportunity he offered me at the beginning of my PhD. To Jo, thank you for your patience and help. To my parents, brothers and sisters, thank you for supporting me throughout these demanding 3 years. Thank you for your endless support and love, I couldn’t have done any of this without you. To the love of my life, Hayoush; you have made my life simply better and remained the motive which kept me going throughout these tough years. Thank you for entering my life at the right time. I would like to thank my friends, Hassan, Khaled and Mohammad as this work would not have been possible without you all. Thanks for all your advice, much appreciated. To Blake, Joanna, Lucy and Sudi, your presence in my life through these tough years has been invaluable. Thanks so much for all the chats we had. Thanks for all the time you spent with me. I would never forget the lunches and coffee breaks we had together. To all my friends in BABS; Anoushcka, Akira, Beth, Chris and Nirmani thank you all for your assistance. I would also like to thank all of my friends who supported me in writing, and incented me to strive towards my goal. vi LIST OF PUBLICATIONS Research articles ñ Review for “Insight into roles of immediate early genes in angiogenesis” (Leonel- Prado-Lourenco, Ahmad M.N. Alhendi, and Levon M. Khachigian). Springer (2014). ñ Transcriptional dynamics implicate non-coding RNAs in the immediate early response. Stuart Aitken, Shigeyuki Magi, Ahmad M.N. Alhendi, Masayoshi Itoh, Hideya Kawaji, Timo Lassmann, Carsten O. Daub, Erik Arner, Piero Carninci, Alistair R.R. Forrest, Yoshihide Hayashizaki, Levon M. Khachigian, Mariko Okada-Hatakeyama and Colin A. Semple. Journal of PLOS Computational Biology (2015). ñ Dynamics of enhancer and promoter activity during mammalian cellular activation and differentiation. Erik Arner &, Carsten O Daub &, Kristoffer Vitting-Seerup &, Robin Andersson &, Berit Lilje, Finn Drablos, Andreas Lennartsson, Michelle Rönnerblad, Olga Hrydziuszsko, Morana Vitezic, Tom C Freeman, Ahmad M.N. Alhendi…el. Journal of Science (2015). ñ Structure & Dynamic of the TnI inhibitory peptide in reconstituted thin filaments as determined by SDSL-EPR. J. Cook, Ahmad M.N. Alhendi, P. Curmi, P. Fajer & L. Brown. In preparation ñ Targeting C-JUN and C-FOS through miR-155 and miR-125b, Potential in combatting melanoma. Ahmad M.N. Alhendi, Leonel-Prado-Lourenco, Noel Whittaker. In preparation Select conference publications ñ Scanning of the inhibitory region of troponin in the muscle thin filament complex. Phani Rekha Potluri, James.A. Cooke, Nicole M. Cordina, Ahmad M.N. Alhendi and Louise J. Brown. Department of Chemistry and Biomolecular Sciences, Macquarie University, Sydney, NSW 2109, Australia. East Coast Protein Meeting 2013. ñ Immediate Early Gene Profiling in Vascular Smooth Muscle Cells Responding to Growth Factor and Cytokine Stimulation. Ahmad M.N. Alhendi, Margaret Patrikakis, Levon M. Khachigian. Centre for Vascular Research, University of New South Wales, New South Wales, Sydney 2052, Australia. Lorne Genome Conference Feb.2014. vii ñ Targeting c-Jun and c-Fos using microRNAs has a potential in contesting melanoma. Ahmad M.N. Alhendi, Leonel Prado-Lourenço, Noel Whitaker. School of Biotechnology and Biomolecular Science, Faculty of Science, University of New South Wales, Sydney, 2052, Australia. American Association of Cancer research conference. April 2015 ñ Novel transcriptional inhibitors for the inhibition of melanoma growth. Taylor, KM; Alhendi, AMN; Halliday, GM; Prado-Lourenço L. Centre for Vascular Research, University of New South Wales, New South Wales, Sydney 2052, Australia. American Association of Cancer research conference. April 2015 viii Table of Contents 1 Introduction .............................................................................................................. 1 1.1 General Introduction Part I: Cardiovascular Disease ............................................ 1 1.1.1 The relevance of the evolution and development of heart to cardiovascular disease 1 1.1.2 History, background and epidemiology of cardiovascular disease ..................... 4 1.1.3 Types of cardiovascular disease ........................................................................... 4 1.1.4 Cardiovascular disease prevention and therapy ................................................. 7 1.1.5 Inflammation and its correlation with atherosclerosis