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The Role of MDM2 and CDK4 in Well Differentiated Liposarcoma Dr The role of MDM2 and CDK4 in well differentiated liposarcoma Dr Rachel Katherine Conyers Submitted in total fulfillment of the requirements of the degree of Doctor of Philosophy April 2015 Department of Pathology The University of Melbourne i Abstract Transformation of normal cells to cancer cells is tightly linked to fundamental changes in cell cycle regulation. In addition, oncogenes can aberrantly enhance cell proliferation. Two genes; Cyclin dependent kinase-4 (CDK4) and Murine double minute 2 (MDM2) are amplified and overexpressed in over 90% of well differentiated liposarcomas. Their role in cell cycle control, and regulation of tumour suppressor p53 respectively, strongly suggesting that deregulation of these genes confers some selective advantage to this tumour. To elucidate the role of these genes in the development and progression of liposarcoma I have used transgenic mouse models and in vitro assays. Given the recent development of novel CDK4 inhibitors, I have tested several CDK4 inhibitors (sc-203873, sc-203874, NPCD, PD 0332991) on liposarcoma cell lines (449B, T1000, 778, GOT3) to determine sensitivity to inhibition, cell cycle arrest and downstream effects of inhibition. PD033991 was found to be the most selective and sensitive CDK4 inhibitor and, as such, was used in a siRNA screen of the genome to identify co-modifiers of CDK4 inhibition. A total of 13 genes were identified that produced a resistance phenotype in the context of CDK4 inhibition. Two of these genes; Arrestin, beta 2 (ARRB2) and Dysferlin (DYSF) demonstrated a reproducible resistance phenotype in a series of functional validation studies. ii Declaration This is to certify that: i the thesis comprises only my original work towards the PhD except where indicated in the Preface, ii due acknowledgement has been made in the text to all other material used, iii the thesis is fewer than 100 000 words in length, exclusive of tables, maps, bibliographies and appendices. __________________________________________________ _____________________ Dr Rachel Katherine Conyers Date iii Preface This is to confirm that all work carried out in this PhD candidature was performed during the time period of the PhD. No work was carried out prior to PhD candidature enrolment. No work has been submitted for other qualifications. The work carried out in this project was all performed by the PhD candidate except for the following: 1. Generation of original MDM2 and CDK4 transgenic mice performed by Ozgene Pty Ltd. 2. Bioinformatics analysis of silencing RNA screen of genome performed by Dr. Kate Gould as part of the Victorian Centre for Functional Genomics (VCFG) at Peter MacCallum Cancer Centre. Editorial assistance was provided by the supervisors of this PhD; Associate Professor David Thomas and Dr. Maya Kansara. Further editorial support was provided by Associate Professor Paul Ekert, Professor Richard Sullivan, Dr. Seong Khaw and Ms Judith Gregory. iv Acknowledgements When I started my PhD, my supervisor, Associate Professor David Thomas, told me thatlike acompleting marathon, aand PhD personally, was like running I think ita marathon.should have He come wasnt with wrong. a government It was much health warning! Having said this, completing my PhD has proven to be one of the most satisfying and fulfilling aspects of my medical career thus far. So the old adage, no pain, no gain, definitely holds true. IdCouncil like to for acknowledge my PhD scholarship. the support I am of grateful the National to both Health my supervisors, and Medical Associate Research Professor David Thomas and Dr Maya Kansara, whose vision created this project, for guiding me through the world of science and teaching me a skill set that I otherwise was not privy too. There are a number of special souls who helped me at various times throughout this journey. Associate Professor Paul Ekert, Dr Seong Khaw deserve special mentions for their help and inspiration at times when I needed it most. To Professor Richard Sullivan and Ms Judith Gregory for their help with the editing process. To my best friends Dr Romi Anaf and Dr Elise Harrison, who must have heard the phrase my PhDour many, at least many, a million years times of friendship. by now, thankTo my you sisters for yourSara unwaveringand Katherine support for always and for enriching my life and being a source of fun and frivolity. To my partner Peter for being a wonderful partner and friend. Finally, I dedicate this PhD to my parents Mr Michael Conyers and Mrs Wendy Conyers. I was lucky enough to have parents who taught me to believe in myself and to strive for whatever I desired in life. My parents taught me that hard work never hurt anybody. They instilled in me both dedication and determination. They are wonderful human beings. Mum and Dad, I love you. v Figures and Tables Chapter 1: Background Figure 1 MDM2 Structure 6 Figure 2 TP 53 Structure 8 Figure 3 Schema of p53 downstream effects 9 Figure 4 Function of CDK4 13 Table 1 Published CDK4/6 inhibitors 25 Table 2 MDM2 inhibitors in clinical trials 27 Chapter 2: Materials and Methods Figure 1 Schematic representation of MDM2 transgene 33 Figure 2 PCR confirming transgene in embryonic stem cells 34 Figure 3 PCR confirming transgene in mice chimera 35 Figure 4 Primer design for mouse studies 37 Figure 5 Experimental design siRNA screen 59 Figure 6 Control setup for high through-put screen 62 Figure 7 Pipeline for determining siRNA screen hits 63 Table 1 DNA lysis mixture 36 Table 2 Primer sequences for mouse studies 37 Table 3 PCR master mix 38 Table 4 Lenti- 40 Table 5 Lenti- n 40 Table 6 PrimerX™ sequences HT Master for Mix RT-PCR Specifications mouse studies 47 Table 7 PrimaryX™ antibodies HT PEI transfectio for Western Blot 51 Table 8 SiRNA smartpool complexes 52 Table 9 Vectors used for MEFS transfections 53 Table 10 NanoJuice Transfection Master Mix 53 Table 11 Antibiotics used in Bacterial Culture 55 Table 12 Master mix for PEI Transfection 55 Table 13 Lenti- 55 Table 14 shRNA vectors 57 Table 15 CDK4 X™inhibitors HT PEI usedtransfection in experiments 58 Table 16 Quality control metrics 64 Chapter 3: Mouse Studies Figure 1 Primer design for Mdm2fl/+ mouse studies 71 vi Figure 2 Genotyping results for mouse studies 71 Figure 3 PCR analysis for Cre-mediated recombination 74 Figure 4 Transfection of Cre-ERT/MDM2fl/+ MEFS. 77 Figure 5 Western blot analysis for MDM2 in Cre-ERT/MDM2fl/+ MEFS 79 Figure 6 Characterisation of the conditional Cre-ERT2/MDM2fl/+ mice 82 Figure 7 GFP detection via immunofluorescence 83 Figure 8 Flow cytometry analyses of reporter GFP expression 85 Figure 9 RT-PCR analysis of Cre-ERT2/MDM2fl/+ mice 87 Figure 10 Western blot analysis for MDM2 in MDM2 mice 88 Figure 11 Stable MEF transfections with CDK4 and MDM2 91 Table 1 Summary of MEF and MDM2fl/+ mouse studies 89 Chapter 4: CDK4 and CDK4 inhibitors in Well differentiated liposarcoma Figure 1 The role of CDK4 in cell cycle progression 96 Figure 2 MTS assay using SC-203874 and SC-203873 on 449B cell line 102 Figure 3 MTS assay using SC-203874 and SC-203873 on 778 cell line 103 Figure 4 CFA using SC-203874 and SC-203873 on 449B cell line 105 Figure 5 MTS assay using NPCD and variety of CDK4 amplified cell lines 107 Figure 6 CFA using NPCD on 449B cell line 108 Figure 7 Western blot analysis of NPCD treated 449B cell line 109 Figure 8 Apoptotic response following NPCD treatment to 449B cell line 111 Figure 9 Western blot of RB1 knockdown in 449B cell line 113 Figure 10 CFA and MTS assay of 449B shRB1 cells treated with NPCD 114 Figure 11 MTS assay of PD03322991 treated melanoma cell lines 115 Figure 12 Western blot of RB1 knockdown in CDK4 amplified cell lines 117 Figure 13 MTS assay performed upon RB1 knockdown cell lines 117 Figure 14 MTS and CFA using PD0332991 on 449B cell lines 119 Figure 15 High dose PD0332991 on 449BshRB1 cell lines 120 Figure 16 Cell cycle profile of PD0332991 treated 449B cell line 121 Figure 17 Western blot analysis of PD0332991 treated 449B cell line 122 Table 1 MTS assay results 101 Chapter 5: Genome wide siRNA screen of the genome to identify co-modifiers of CDK4 inhibition in Well differentiated liposarcoma Figure 1 Optimisation workup for siRNA screen 134 Figure 2 Pipeline for siRNA screen 136 Figure 3 Optimisation and experimental design 138 Figure 4 Optimisation of transfection based on RB1 silencing 142 Figure 5 CTG signal stability over extended timeframe 143 Figure 6 Optimisation of transfection controls 146 Figure 7 Quality control measures for screen (Box plots) 150 vii Figure 8 Well scatter plots of optimisation plates 152 Figure 9 Repeat optimisation experiments: effects on quality control 153 Figure 10 Well scatter plots of repeat optimisation plates 154 Figure 11 Pipeline for determining screen resistance hits 156 Figure 12 Well scatter plots of primary screen 158 Figure 13 Quality control metrics of secondary screen 162 Figure 14 Suppression of RNA expression of siRNA targets 169 Figure 15 Tertiary screen results across CDK4 amplified cell lines 170 Figure 16 Validation studies of final screen hits 173 Table 1 Transfection reagents and efficiency 139 Table 2 Transfection reagent conditions using PLK1 and siGLO 140 Table 3 Efficiency of gene silencing by RT-PCR 141 Table 4 Quality control measures used in screen 148 Table 5 Primary screen quality control metrics 157 Table 6 Validated resistance hits for secondary screen 161 Table 7 GSEA analysis of secondary screen siRNA targets 165 Table 8 GeneGo analysis of secondary screen siRNA targets 166 Table 9 Gene list for tertiary
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