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(Title of the Thesis)* TARGETING PROSTATE CANCER BY SMALL MOLECULES by JIAN ZHANG Submitted in partial fulfillment of the requirements For the degree of Doctor of Philosophy Dissertation Adviser: Dr. Andrei Gudkov Department of Biochemistry CASE WESTERN RESERVE UNIVERSITY May, 2011 CASE WESTERN RESERVE UNIVERSITY SCHOOL OF GRADUATE STUDIES We hereby approve the dissertation of Jian Zhang candidate for the Doctor of Philosophy degree *. (signed) David Samols (chair of the committee) Andrei V. Gudkov Donal S. Luse Hung-Ying Kao Julia V. Kichina Warren D. Heston (date) August 24, 2010 *We also certify that written approval has been obtained for any proprietary material contained therein. ii Table of Contents List of Tables ..................................................................................................................... vi List of Figures ................................................................................................................... vii Acknowledgements ............................................................................................................ ix List of Abbreviations .......................................................................................................... x Abstract ............................................................................................................................ xiv Chapter 1. Introduction ....................................................................................................... 1 1.1. The Prostate and Prostate Cancer ............................................................................. 1 1.1.1. Prostate .............................................................................................................. 1 1.1.2. Androgen Receptor ............................................................................................ 5 1.1.3. Prostate Cancer .................................................................................................. 9 1.2. Treatment of Prostate Cancer ................................................................................. 11 1.2.1. Hormone Therapy ............................................................................................ 14 1.2.2. AR in CRPCa from Hormone Therapy ........................................................... 18 1.2.3. Palliative Therapies ......................................................................................... 21 1.3. Experimental Plan and General Strategy of Studies .............................................. 24 1.4. Preliminary Data 1: Screening of Small Molecule Library .................................... 29 1.4.1. Rationale and Approach .................................................................................. 29 1.4.2. High Throughput Screening ............................................................................ 32 1.4.3. Long-term Survival Assay ............................................................................... 38 1.4.4. Summary.......................................................................................................... 41 1.5. Preliminary Data 2: In Vivo Evaluation Therapeutic Efficiency of 5582 .............. 42 Chapter 2. Materials and Methods .................................................................................... 44 2.1. Material .................................................................................................................. 44 2.1.1. Cell Lines......................................................................................................... 44 2.1.2. Chemicals ........................................................................................................ 47 2.1.3. Plasmids ........................................................................................................... 48 2.1.4. PCR Primers .................................................................................................... 49 2.1.5. Solutions .......................................................................................................... 49 iii 2.2. Method ................................................................................................................... 53 2.2.1. Cell Survival Assay ......................................................................................... 53 2.2.2. Flow Cytometric Assay ................................................................................... 54 2.2.3. Hybridization Assays....................................................................................... 55 2.2.4. Immunostaining ............................................................................................... 56 2.2.5. Nucleic Acids Preparation ............................................................................... 57 2.2.6. Polymerase Chain Reaction (PCR) ................................................................. 59 2.2.7. Transfection ..................................................................................................... 61 2.2.8. Reporter Assay ................................................................................................ 62 2.2.9. RNA Interference Analysis ............................................................................. 63 2.2.10. Western blotting Analysis ............................................................................. 63 2.2.11. Lentivirus Viral Transduction ....................................................................... 64 2.2.12. In vivo Evaluation Therapeutic Potential of ARKILs ................................... 65 Chapter 3. Approaching Mechanisms of the Hits Specific Cytotoxicity .......................... 67 3.1. Hypothesis, Rationale and Approaches .................................................................. 67 3.2. Characterization of the Hits by Cytotoxicity Assay ............................................... 69 3.3. Mechanism of Prostate Cell Death ......................................................................... 76 3.4. Are AR Involved in the Isolated Hits Cytotoxicity? .............................................. 79 3.4.1. Effect of the Hits on AR Transactivation Function in Sensitive PCa Cells .... 79 3.4.2. Do the Isolated Compounds Affect AR Protein Abundance? ......................... 85 3.4.3. The Isolated Hits were Named as ARKILs ..................................................... 87 3.5. Is AR a Potential Target of the Isolated Hits ......................................................... 89 3.5.1. Potential mechanism of ARKILs..................................................................... 89 3.5.2. Is Translocation of AR Affected by ARKILs? ................................................ 91 3.5.3. Do ARKILs Influence the Stability of AR and AR mRNA? .......................... 94 3.5.4. Is AR Itself Related to ARKILs Toxicity? ...................................................... 97 3.5.5. Summary of the AR Biased Investigations ................................................... 100 3.6. Identification and Characterization of ARKIL Resistant Variants of 22Rv1 ...... 102 3.6.1. Isolation of Resistant Variants....................................................................... 102 3.6.2. Are Resistant Variants Cross-Resistant to ALL ARKILs? ........................... 109 3.6.3. Non-involvement of Multidrug Resistance Transporters .............................. 116 iv 3.6.4. Characterization of ARKIL Resistance in AKRVs. ...................................... 120 3.7. Summary .............................................................................................................. 130 Chapter 4. Investigating Potential Target of ARKILs .................................................... 130 4.1 Gene Profiling Analysis on AKRVs and Parental 22Rv1 cells ............................ 131 4.1.1 Hypothesis and Background ........................................................................... 131 4.1.2 Annotated DEGs from Microarray Analysis .................................................. 134 4.1.3 Validation of c-Maf Involvement in ARKIL-3 toxicity ................................. 143 4.2 DEGs Validation by shRNA Library High Throughput Screening ...................... 155 4.3 Summary ............................................................................................................... 159 Chapter 5. In vivo Evaluation of Therapeutic Potential .................................................. 160 5.1. Pilot Toxicity Assessment of ARKILS ................................................................ 160 5.2. Evaluation of in vivo Efficacy of ARKIL-3 ......................................................... 165 5.3. Summary .............................................................................................................. 168 Chapter 6. Conclusions and Discussion .......................................................................... 169 6.1. Conclusions .......................................................................................................... 169 6.2. Discussion ............................................................................................................ 171 6.3. Working Model .................................................................................................... 180 Biobliography ................................................................................................................. 182 v List of Tables Table 1-1. Treatments for prostate cancer .......................................................................
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