Unraveling of the Major Genetic Defects in Prostate Cancer

Unraveling of the major genetic defects in prostate cancer Karin Hermans The studies described in this thesis were performed at the Department of Pathology of the Josephine Nefkens Institute, Erasmus MC, Rotterdam, The Netherlands Layout and printing by Optima Grafische Communicatie, Rotterdam Publication of this thesis was financially supported by the Department of Pathology of the Erasmus MC, Novartis, and the Erasmus University Rotterdam Cover: 3D culture of immortalized benign prostate epithelial cells (PNT2C2) infected with lentivirus expressing green fluorescent protein. © 2008 K.G.L. Hermans No part of this thesis may be reproduced, stored in a retrieval system or transmitted in any form or by means, electronical,mechanical, photocopying, recording or otherwise, without written permission of the author. Several chapters are based on published papers, which were reproduced with permission of the co-authors. Copyright of these papers remains with the publisher. Unraveling of the major genetic defects in prostate cancer Ontrafelen van de meest belangrijke genetische defecten in prostaatkanker Proefschrift Ter verkrijging van de graad van doctor aan de Erasmus Universiteit Rotterdam op gezag van de rector magnificus Prof. Dr. S.W.J. Lamberts en volgens besluit van het College voor Promoties De openbare verdediging zal plaatsvinden op woensdag 28 januari 2009 om 11.45 uur door Karin Gertruda Louisa Hermans Geboren te Boxmeer PROMOTIECOMMISSIE Promotor: Prof.dr.ir. J. Trapman Overige leden: Dr.ir. G. Jenster Prof.dr. A. Geurts van Kessel Prof.dr. J.W. Oosterhuis Contents List of abbreviations 6 Chapter 1 General introduction 7 Chapter 2 Array-based comparative genomic hybridization guides 33 identification of N-COR as a novel tumor suppressor gene in prostate cancer Chapter 3 Loss of a small region around the PTEN locus is a major 59 chromosome 10 alteration in prostate cancer xenografts and cell lines Chapter 4 TMPRSS2:ERG fusion by translocation or interstitial deletion is 83 highly relevant in androgen-dependent prostate cancer, but is bypassed in late stage androgen receptor negative prostate cancer Chapter 5 Overexpression of prostate-specific TMPRSS2(exon 0)-ERG fusion 99 transcripts corresponds with favorable prognosis of prostate cancer Chapter 6 Two unique prostate-specific and androgen-regulated fusion 115 partners of ETV4 in prostate cancer Chapter 7 Truncated ETV1, fused to novel tissue-specific genes, and full 129 length ETV1 in prostate cancer Chapter 8 General discussion 153 Summary 167 Samenvatting 169 Curriculum Vitae 171 List of publications 173 Dankwoord 175 List of abbreviations AR androgen receptor MKK4 mitogen-activated protein BAC bacterial artificial chromo- kinase kinase 4 some MMP matrix metalloproteinase BPH benign prostatic hyperplasia N-COR nuclear corepressor CANT1 calcium activated nucleoti- NP normal prostate dase 1 PBGD porphobilinogen deaminase CGH comparative genomic PCR polymerase chain reaction hybridization PI3K phosphatidylinositol 3-kinase COPA cancer outlier profile analysis PIN prostate inter-epithelial DHT dihydrotestosterone neoplasia ERG ETS Related Gene PTEN phosphatase and tensin ETS E26 Transforming Sequence homologue deleted on ETV1 ETS variant gene 1 chromosome 10 ETV4 ETS variant gene 4 PSA prostate specific antigen ETV5 ETS variant gene 5 QPCR quantitative polymerase EST expressed sequence tag chain reaction FISH fluorescent in situ hybridiza- RLM-RACE RNA ligase mediated rapid tion amplification of cDNA ends FOXA1 forkhead box A1 RP radical prostatectomy FOXP1 forkhead box P1 RT-PCR reverse transcriptase poly- GFP green fluorescent protein merase chain reaction HD homozygous deletion TAD transactivation domain HERV human endogenous retrovi- TMPRSS2 transmembrane protease, rus serine 2 KLK2 kallikrein 2 TURP transuretheral resection of LOH loss of heterozygosity the prostate LTR long terminal repeat siRNA small interfering RNA MIPOL1 miror-image polydactyly 1 SNP single nucleotide polymor- phism Chapter 1 General Introduction General Introduction PROSTATE CANCER In developed countries, prostate cancer is the most common malignancy in men and a major cause of cancer-related death (1). In The Netherlands 93 new cases per 100,000 men were detected in 2003 (The Netherlands Cancer Registry). Prostate cancer incidence varies between different ethnic groups. African-American men have the highest incidence rates, followed by Caucasian-American men and men in Western Europe and Australia. The lowest incidence rates are observed in the Asian population. These differences may be explained by environmental and dietary factors. However, genetic factors, i.e. polymorphisms in genes that can predispose to cancer, may also play a role in prostate cancer development. A major risk factor of prostate cancer is age, since it is predominantly a disease of the senior adult (i.e. men over the age of 65 years). Below the age of 55 the incidence of prostate cancer is very low (2, 3). During recent years our knowledge of the molecular mechanism underlying prostate cancer development and progression has rapidly increased. However, there are still many gaps in our knowledge that remain to be filled. Genetic analyses of different stages of prostate cancer will increase our knowledge of the molecular background of the development and progression of prostate cancer. The identification of novel biomarkers will help to predict the clinical course of the disease. Furthermore, a better understanding of the molecular mechanism of prostate tumorigenesis is essential for development of novel targeted therapies. Androgen signalling mediated by the androgen receptor (AR) is essential for development and maintenance of the normal prostate (4). The majority of prostate cancers also depend on a functional AR. The AR is a member of the steroid hormone receptor transcription factor family. It is activated by binding of androgens (testosterone or dihydrotestosterone (DHT)). The AR regulates the transcription of many target genes by binding to androgen response elements in the promoter or enhancer regions. The ARHoofdstuk recruits specific 1 cofactors to either activate or repress a specific target gene. In the normal prostate there is a balance of expression of androgen-regulated genes involved in differentiation, proliferation, function and survival. This balance is moved towards proliferation and survival in prostate cancer cells (Figure 1). Figure 1. Model of androgen receptor function in the normal adult prostate (A) and in prostate cancer (B) 9 Figuur 1 Chapter 1 Figure 2. Overview of prostate cancer development and progression. A well-known androgen-regulated and prostate-specific gene is Prostate Specific Antigen (PSA). The PSA protein is secreted by the prostate. In prostate cancer patients PSA levels can be measured in serum. Elevated serum PSA levels are a first indication of prostate cancer, however, elevated PSA might also be caused by benign prostatic Figuurhyperplasia 2 (BPH) or prostatitis. Introduction of the PSA test in the 1980’s has increased prostate cancer incidence considerably. However, a proportion of the detected cancers will never become life threatening. Consecutive procedures currently used for prostate cancer diagnosis include: serum PSA detection, digital rectal examination, transrectal ultrasonography and histopathological examination of biopsy specimens (5). The histo- logical grade, the tumour stage and PSA level at diagnosis are used to predict prostate cancer progression. Yet, at present, it is impossible to accurately discriminate between patients with clinically significant and more indolent disease (6). In Figure 2, a schematic representation of the different stages of prostate cancer development and progression is depicted. Prostatic intra-epithelial neoplasia (PIN) is considered to represent the precursor lesion of prostate cancer, because similar genetic alterations are found both in PIN and in primary prostate cancer. Tumours confined to the prostate can either be treated by surgical removal of the prostate or by local radiation therapy. A third option is active surveillance, i.e. a patient is not treated, but instead is closely monitored for disease progression. Because the AR plays a critical role in overall function of the normal prostate as well as in growth and survival of malignant prostate cells, primary therapy of metastatic disease is androgen-deprivation (endocrine therapy). This therapy is based on inhibition of AR transcriptional activity, by either inhibiting androgen production or by blocking AR function with anti-androgens, such as hydroxy-flutamide or bicalutamide. Initially endocrine therapy results in regres- sion of the tumour. However, eventually all tumours become resistant to hormonal abla- tion therapy and progress to androgen-independent disease. In most (> 80%) hormone refractory tumours the AR is still expressed and still plays a role in the disease (7). In 10 General Introduction part of these tumours the AR gene is amplified and the protein over expressed (8). In a small percentage the AR is mutated (9), turning antagonists into agonists. Other factors that may contribute to an active AR in endocrine resistant prostate cancers are over expression of cofactors or inhibition of corepressors (10). Recently, evidence has been provided that expression of enzymes that convert adrenal androgens to DHT or even synthesize DHT from cholesterol, is induced in prostate cancer (11). In a small proportion of prostate cancers the AR is bypassed

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