ALFONSO URBANUCCI Overexpression of Androgen Receptor in Prostate Cancer ACADEMIC DISSERTATION To be presented, with the permission of the board of Institute of Biomedical Technology of the University of Tampere, for public discussion in the Jarmo Visakorpi Auditorium, of the Arvo Building, Lääkärinkatu 1, Tampere, on January 20th, 2012, at 12 o’clock. UNIVERSITY OF TAMPERE ACADEMIC DISSERTATION University of Tampere, Institute of Biomedical Technology and BioMediTech Tampere University Hospital, Laboratory Centre Graduate Program in Biomedicine and Biotechnology (TGPBB) Finland Supervised by Reviewed by Professor Tapio Visakorpi Docent Auli Karhu University of Tampere University of Helsinki Finland Finland Docent Noora Kotaja University of Turku Finland Copyright ©2012 Tampere University Press and the author Distribution Tel. +358 40 190 9800 Bookshop TAJU Fax +358 3 3551 7685 P.O. Box 617 [email protected] 33014 University of Tampere www.uta.fi/taju Finland http://granum.uta.fi Cover design by Mikko Reinikka Acta Universitatis Tamperensis 1693 Acta Electronica Universitatis Tamperensis 1159 ISBN 978-951-44-8685-2 (print) ISBN 978-951-44-8686-9 (pdf) ISSN-L 1455-1616 ISSN 1456-954X ISSN 1455-1616 http://acta.uta.fi Tampereen Yliopistopaino Oy – Juvenes Print Tampere 2012 CONTENTS ABBREVIATIONS ..................................................................................................... 5 ABSTRACT ................................................................................................................ 7 SINTESI ...................................................................................................................... 8 INTRODUCTION ..................................................................................................... 10 LIST OF ORIGINAL COMMUNICATIONS .......................................................... 12 1. REVIEW OF THE LITERATURE .................................................................... 13 1.1 Prostate cancer ................................................................................................. 14 1.1.1 Natural history of prostate cancer ............................................................. 14 1.1.2 Androgen dependency and treatment options for prostate cancer ............ 15 1.1.3 Castration-resistant prostate cancer and treatment options ...................... 16 1.1.4 Molecular pathology of prostate cancer ................................................... 18 1.1.4.1 PC susceptibility ................................................................................ 18 1.1.4.2 GSTP1 ............................................................................................... 18 1.1.4.3 NKX3.1 ............................................................................................. 18 1.1.4.4 ETS rearrangement ............................................................................ 19 1.1.4.5 PTEN ................................................................................................. 20 1.1.4.6 TP53 .................................................................................................. 21 1.1.4.7 RAS/RAF signaling ........................................................................... 22 1.1.4.8 RB1 .................................................................................................... 22 1.1.4.9 Non-coding RNAs ............................................................................. 22 1.1.4.10 Amplification and overexpression of MYC ...................................... 23 1.2 Androgen receptor-mediated mechanisms ...................................................... 24 1.2.1 Androgen receptor: structure and function ............................................... 24 1.2.2 Androgen receptor and castration-resistant prostate cancer ..................... 25 1.2.2.1 Androgen receptor overexpression .................................................... 26 1.2.2.2 Steroidogenesis .................................................................................. 27 1.2.2.3 Androgen receptor mutations ............................................................ 29 1.2.2.4 Androgen receptor interacting proteins ............................................. 29 1.2.2.4.1 Components of the general transcription machinery .................. 30 1.2.2.4.2 Coregulators modifying chromatin status .................................. 30 1.2.2.4.3 Coregulators with other functions .............................................. 33 1.2.2.5 Androgen receptor coregulators and prostate cancer ........................ 35 1.2.3 Ligand independent activation of the androgen receptor ......................... 36 1.2.3.1 Androgen receptor variants ............................................................... 37 1.2.4 Androgen receptor-mediated transcription ............................................... 40 1.2.4.1 Formation of the transcription initiation complex ............................. 40 1.2.4.1.1 Androgen responsive elements ................................................... 42 1.2.4.2 Modulation of androgen receptor transcriptional activity by other transcription factors ........................................................................... 43 1.2.5 Androgen receptor target genes and prostate cancer ................................ 44 2. AIMS OF THE STUDY .................................................................................... 46 3 3. MATERIALS AND METHODS ....................................................................... 47 3.1 Cell lines and cell culture procedure (Studies I-III) ........................................ 47 3.2 Xenografts material (Study I) .......................................................................... 48 3.3 Clinical samples (Study I) ............................................................................... 48 3.4 Chromatin immunoprecipitation (Studies I and II) ......................................... 48 3.5 ChIP-seq assays (Study I) ................................................................................ 48 3.6 ChIP-seq data analysis, Motif overrepresentation analysis, Gene Ontology analysis and mRNA expression profiling (Study I) ........................................ 49 3.7 qRT-PCR (Studies I-III) .................................................................................. 49 3.8 ChIPqPCR (Studies I and II) ........................................................................... 50 3.9 Statistical analysis (Studies I-III) .................................................................... 50 3.10 Western blot (Studies I and III) ....................................................................... 51 3.11 Immunohistochemistry (Study I) ..................................................................... 51 3.12 siRNA transfections and Growth curves (Study I) .......................................... 51 4. RESULTS AND DISCUSSIONS ...................................................................... 53 4.1 AR overexpression enhances AR mediated signaling (Studies I-III). ............. 53 4.1.1 Binding of AR to chromatin in low androgen concentration is enhanced in AR-overexpressing cells (Study I) ........................................................... 53 4.1.2 AR binding occurs earlier and more powerfully in AR-overexpressing cells grown in low concentrations of androgens (Study II) ...................... 56 4.1.3 Chromatin is more open in AR-overexpressing cells (Study II) .............. 58 4.1.4 AR overexpression enhances androgen regulation of AR coactivators (Studies I and III) ..................................................................................... 60 4.2 Identification of AR target genes in PC (Study I) ........................................... 62 4.2.1 Gene ontology .......................................................................................... 62 4.2.2 AR target genes identified solely using ARBSs data (unpublished data) 63 4.2.3 AR target genes identified using ARBSs and microarray expression data (Study I) .................................................................................................... 64 4.3 Validation and characterization of AR target genes and significance in PC (Study I) ........................................................................................................... 67 4.3.1 Androgen regulation and expression in clinical material ......................... 67 4.3.2 ARBSs validation (Study I) ...................................................................... 69 4.3.3 Functional significance of FEN1, ZWINT and SNAI2 for PC cells growth (Study I) .................................................................................................... 70 4.3.4 FEN1 expression predicts clinical outcome ............................................. 70 5. CONCLUDING REMARKS ............................................................................. 72 6. REFERENCES .................................................................................................. 74 Acknowledgements ................................................................................................... 94 ORIGINAL COMMUNICATIONS ......................................................................... 96 4 ABBREVIATIONS AcH3 acetylation of histone 3/acetylated histone 3 AD androstenedione ADT androgen deprivation therapy AF1 activation function 1 AF2 activation function 2 AKT Official name: AKT1 (v-akt murine thymoma viral oncogene homolog 1) ANOVA analysis of variance AR