Targeting the Transcription Factor c-Jun in Cervical Cancer Cells Grace Pei Chien Yee Centre for Vascular Research, School of Medical Sciences, University of New South Wales, Australia A thesis submitted to the University of New South Wales for the degree of Doctor of Philosophy (PhD) September 2013 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 …………………………………………….............. i ABSTRACT Despite the development of vaccines for human papillomaviruses (HPV) in cervical cancer and other efforts to improve therapy, deaths still average 275,000 annually worldwide, with most women succumbing to recurrent or metastatic disease. The c-Jun oncogene is a subunit of the activating protein-1 (AP-1) transcription factor and is strongly expressed in cervical cancer, regulating the expression of HPV16 and 18 genes. AP-1 plays a major role in cell growth, migration and apoptosis in many cell types. This work examined the role of c-Jun in modulating cervical cancer cell line (HeLa) proliferation, migration, apoptosis, invasion, susceptibility to cisplatin and the underlying mechanisms. c-Jun protein and mRNA levels were reduced by c-Jun siRNA. c-Jun silencing inhibited cell proliferation. Significantly, c-Jun suppression dramatically reduced HeLa migration and invasion and targeted down-regulation of cyclooxygenase-2 (Cox-2), intracellular adhesion molecule 1 ( ICAM-1), matrix metalloproteinases (MMP)-1 and -9 genes highly expressed in cervical cancer and associated with metastatic growth. siRNA knockdown of Cox-2 also reduced HeLa migration and invasion as well as MMP-1 expression suggesting an intermediary link. In transfected cells over-expressing c-Jun, cell proliferation was not significantly increased but cell invasiveness was markedly enhanced in parallel with enhanced Cox-2 and MMP-1 expression as well as MMP-2 activity. Modulation of c-Jun expression did not synergistically combine with cisplatin to increase the susceptibility of HeLa cells to apoptosis or cell cycle disruption. In vivo, c-Jun siRNA pre-transfected HeLa-luc solid tumor growth and size were significantly retarded compared to the control groups. siRNA targeting another transcription factor, ii Early Growth Response-1 (Egr-1) demonstrated significant inhibition of HeLa cell migration and invasion as well as reduced MMP-1 expression and MMP-9 activity, with concomitant blockade of c-Jun and Cox-2 expression, suggesting pivotal link of these genes to HeLa cell migration and invasion. Reduced invasion potential of HeLa cells after c-Jun, Egr-1 and Cox-2 knockdown, respectively suggests the potential of these genes as targets in treatment of metastatic and recurrent cervical cancer. Data also suggest a m echanism involving c-Jun, Egr-1 and Cox-2 in the regulation of MMP-1. iii PUBLICATIONS AND CONFERENCE PRESENTATIONS Publications Grace Pei Chien Yee, Paul de Souza, Levon M. Khachigian. Current and Potential Treatments for Cervical Cancer. Current Cancer Drug Targets. 2013 Feb;13(2):205- 20 Grace Pei Chien Yee, Paul L. De Souza, and Levon M. Khachigian. Reducing invasion potential of cervical cancer cells via targeted knockdown of c-Jun. ASCO MEETING ABSTRACTS Jun 17, 2013:e22005 Conference Presentations 1. May 2013, “c-Jun silencing reduces the invasion potential of cervical cancer cells”, poster in 2nd Lowy Cancer Symposium, Sydney. 2. September 2012, “Silencing the transcription factor c-Jun in cervical cancer”, oral presentation in Centre for Vascular Research Symposium, Sydney. 3. September 2012, “Silencing the transcription factor c-Jun in cervical cancer cells”, poster in Australian Vascular Biology Society Scientific Meeting, Queensland. 4. June 2012, “Targeting the transcription factor c-Jun in cervical cancer cells”, poster in the Australian Society for Medical Research, 20th NSW Scientific Meeting, Sydney. Award Runner-up for best poster presentation, “Silencing the transcription factor c-Jun in cervical cancer cells”, poster in Australian Vascular Biology Society Scientific Meeting, Queensland, September 2012. iv ACKNOWLEDGEMENTS I would like to thank my supervisor, Professor Levon Khachigian and co-supervisor, Professor Paul de Souza, for their guidance and support throughout my PhD studies. I would also like to thank all past and present members of the Khachigian lab, especially to Dr. Fernando Santiago, for mentoring me at the beginning of my PhD studies; to Dr. Lucinda McRobb, for peer reviewing my thesis and intellectual contribution to my work; to Dr. Leonel Prado-Lourenco, for his guidance in animal work and always willing to answer my many questions; and to Margaret, for her moral support especially during the beginning of my motherhood. Thank you again for all the support and encouragement particularly during my hard time. I would also like to thank those who helped me with the work related to this thesis, especially to Professor Wendy Jessup from ANZAC Research Institute, for her kind gift of HeLa cells; Dr Maaike Kockx from ANZAC Research Institute, for her advice and positive control for zymography; and to Fei Shang from the Histology and Microscopy Unit, for her help with immunohistochemistry. Last but not least, I would like to thank my dearest family: Dad, Seng Hoy, Mum, Lee Lee, Alex and Richard for always giving me endless love and support throughout my life; to my husband, Henry, for his great understanding and effort in sharing the household chores throughout my PhD; to my daughter, Claire, for being a good girl most of the time. I love you all. v LIST OF FIGURES Figure 1.1: The cervical transformation zone ------------------------------------- 4 Figure 1.2: Schematic image of HPV 16 genome and integration into host chromosome ------------------------------------------------------- 9 Figure 1.3: The HPV life cycle ----------------------------------------------------- 10 Figure 1.4: HPV E6 and E7 regulated pathways and genes --------------------- 12 Figure 1.5: Current treatments for cervical cancer ------------------------------ 18 Figure 1.6: Potential future therapy for cervical cancer ---------------------- 22 Figure 1.7: Schematic representation of the antigene or antisense approach ---- 26 Figure 1.8: Schematic representation of the ribozyme or DNAzyme approach -- 29 Figure 1.9: Schematic representation of the aptamer approach -------------- 30 Figure 1.10: Schematic representation of the siRNA approach -------------- 31 Figure 1.11: Mechanisms which enable rapid accumulation of IEGs -------------- 44 Figure 1.12: Formation of AP-1 transcription factor from c-Jun and c-Fos heterodimerization -------------------------------------------------- 46 Figure1.13: Regulation of c-Jun transcriptional activity by de-repression model -------------------------------------------------------------------- 47 Figure 1.14: The effects of c-Jun in apoptosis -------------------------------- 50 Figure 3.1: Molecular mechanisms of cell migration ----------------------- 85 Figure 3.2: Serum induces the expression of c-Jun in HeLa cells -------------- 89-90 Figure 3.3: Uptake of FITC-siRNA by HeLa cells -------------------------------- 91-92 Figure 3.4: c-Jun-targeting siRNA inhibits HeLa c-Jun mRNA & protein expression --------------------------------------------------------------- 93-94 Figure 3.5: c-Jun siRNA reduces HeLa cell proliferation ----------------------- 95 Figure 3.6: c-Jun siRNA inhibits HeLa cell migration ----------------------- 96-97 Figure 3.7: c-Jun siRNA inhibits HeLa cell invasion -------------------------------- 98-99 vi Figure 3.8: c-Jun siRNA inhibits HeLa cell migration and regrowth in a scratch wound assay --------------------------------------------- 100-101 Figure 3.9: c-Jun siRNA downregulates mRNA levels of c-Jun target genes in HeLa cells ----------------------------------------------------- 103-104 Figure 3.10: c-Jun siRNA downregulates the protein expression of Cox-2 but does not significantly inhibit ICAM-1 ----------------------- 105 Figure 3.11 c-Jun siRNA does not inhibit the activity of MMP-1, MMP-2 or MMP-9 in HeLa cells -------------------------------------- 107-108 Figure 3.12: c-Jun siRNA inhibits HPV18 E6 and HPV18 E7 mRNA expression -109 Figure 3.13: Serum inducibility of Cox-2 mRNA and protein expression ------- 111-112 Figure 3.14 Cox-2 siRNA inhibits Cox-2 mRNA and protein expression in HeLa cells ---------------------------------------------------------- 113-114 Figure 3.15: Cox-2 siRNA significantly inhibits HeLa cell migration and invasion ----------------------------------------------------------- 115-116 Figure 3.16: Cox-2 siRNA reduces MMP1 mRNA expression in HeLa cells --- 117 Figure 3.17: Cox-2 siRNA does not inhibit MMP-1, MMP-2 and MMP-9 activities --------------------------------- 118-119 Figure 3.18 Uptake of GFP plasmid by HeLa cells