Nigel Bennett (2010). ANDROGEN RECEPTOR, CAVEOLIN-1 AND ANDROGEN SELF-SUFFICIENCY IN PROSTATE CANCER PhD Thesis, School of Medicine, The University of Queensland.

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Author Nigel Bennett
School, Centre or Institute School of Medicine
Institution The University of Queensland
Publication date 2010-03
Thesis type PhD Thesis
Total pages 297
Total colour pages 38
Total black and white pages 259
Subjects 11 Medical and Health Sciences
Abstract/Summary Prostate cancer (PCa) is the third most common cancer and the second leading cause of male deaths in Western countries. Radical prostatectomy is suitable for treatment of early clinical stage PCa that is confined to the gland. More advanced clinical stage disease, particularly with metastases, is treated with androgen deprivation or blockade therapies. Tumours that recur following these forms of therapy are often referred to as castrate-resistant. Castrate-resistant PCa (CRPC) has a poor patient prognosis, with patient death occurring within five years. The castrate-resistant phenotype is thought to develop because of selection pressures by androgen deprivation therapies (ADT) that induce altered expression and activation of many proteins. The protein target for ADT is a steroid nuclear receptor called androgen receptor (AR). In normal prostatic epithelial cells, AR expression, activity and the associated tissue maintenance are regulated by the androgens dihydrotestosterone (DHT) and testosterone. AR interacts with many co-regulator proteins that function to either co-activate or co-repress its activation. In PCa, the membrane protein Caveolin-1 (Cav-1) is considered to be an AR co-activator protein. Cav-1 is not found in normal prostatic glandular epithelium, the origin for the majority of prostatic adenocarcinomas. Cav-1 expression and prevalence increases with PCa progression, in many cases correlating positively with tumour stage and grade. Cav-1 is localised to caveolae, small plasma membrane invaginations that have roles in cell trafficking, endocytosis and signalling. Caveolae formation, structural maintenance and signalling rely heavily upon the sterol compound cholesterol which is abundant in the plasma membrane of cells. Cholesterol is also closely integrated with androgen signalling and is the precursor compound for steroidogenesis leading to androgen synthesis. Although via completely different mechanisms, the intimate relationship of cholesterol with AR and Cav-1 appears necessary for their transient plasma membrane interactions. This molecular “ménage à trios” provides scope for investigating multiple therapeutic targets, both internally and peripherally to this trio, which may prove beneficial to PCa sufferers. This thesis focused on AR, its co-regulation by Cav-1, and the involvement of cholesterol and steroidogenic potential to enhance AR activity, with the potential to use this information for development of novel CRPC therapies. Chapter 1 provides a review of the literature. Chapter 2 describes the general Materials and Methods used during this project. Specific methods are also described in each of the research Chapters (Chapters 3 to 6). Chapter 7 gives an overview of the results and presents future directions for the project. Chapter 1 (Literature Review) includes segments on the molecular cell biology of AR and the known interactions between AR, Cav-1 and cholesterol metabolism. The contents of this Chapter have been published in two first-author reviews by the PhD candidate. • Chapter 1 developed the overall thesis hypotheses: o that there are mechanisms that determine regulation of AR-mediated transcription involving Cav-1 that differ depending on the castrate-resistant state of PCa; o that modulation of Cav-1 or AR by various strategies will show a functional link between Cav-1 and AR; o that AR, Cav-1 and cholesterol metabolism are linked in PCa via steroidogenic pathways; o and that mechanisms determined in vitro will be also found using tissue from human benign prostatic hyperplasia and PCa of different Gleason scores and cancer grades. • The specific aims of the thesis project were: o to investigate the relative expression profiles of Cav-1 and AR in the range of selected PCa and non-PCa cell lines; o to use AR inhibition or activation strategies to investigate whether the interaction in these cell lines between Cav-1 and AR is androgen-dependent; o to determine whether the removal or modulated expression of Cav-1 or AR affects the activity of the other; to determine the role that cholesterol and steroidogenesis play in the interrelationship between Cav-1 and AR and the development of PCa; o and to use PCa, benign prostatic hyperplasia (BPH), and normal prostatic tissue from humans to investigate the localisation and levels of expression of AR, Cav-1 and a panel of steroidogenic enzymes compared with cancer grade. In Chapter 3, the PCa cell line-specific signatures of AR and Cav-1 were investigated using mRNA, protein, and reporter assay analyses. The experiments were designed to ask if there is a link between these patterns and the development of castrate resistance. AR and Cav-1 expression was compared in six PCa cell lines of known levels of androgen sensitivity: LNCaP cells express AR and are androgen-dependent; 22Rv1 cells express AR, but are not androgen-dependent. 22Rv1 cells can survive without androgens but will respond positively when treated with androgens thus they are considered androgen responsive; PC3 and DU145 cells do not express AR and are not androgen-dependent or responsive, representing castrate-resistant disease; ALVA41 cells express AR and are androgen-responsive but not dependent upon androgen for survival; and RWPE1 cells were derived from normal prostate tissue and have been immortalised for long-term culture. The cellular changes and AR or Cav-1 expression in these cell lines after AR or Cav-1 modulation were also analysed. AR expressing cell lines LNCaP (androgen sensitive) and 22Rv1 (androgen responsive) both had low levels of Cav-1. Cell lines that did not express AR protein in our investigations (DU145, PC3, ALVA41 and RWPE1) all had high levels of Cav-1. When caveolae were disrupted there was little change to cell viability, but Cav-1 knock down diminished AR expression. When AR expression was knocked down, Cav-1 expression was also diminished. These data suggest that Cav-1 expression in AR positive cell lines is regulated, at least partially, by AR and loss of AR has a deregulatory effect upon Cav-1 expression. The grade and stage of the cancer may be important: Cav-1 expression may be regulated in early grade/stage PCa but be deregulated by AR loss in late grade/stage tumours as evidenced by high Cav-1 expression in AR negative cell lines. In Chapter 4, the localisation and intensity of expression of AR and Cav-1 were investigated in sixty-eight prostate tissue samples: normal (2), BPH (6) and cancerous tissues (62 of varying Gleason scores) using immunohistochemistry. The primary cells of interest during this investigation were the glandular epithelium, which are the cells of origin for the majority of prostatic adenocarcinomas, and the supporting basal cell layer, and to a lesser extent, the stromal tissue. AR was found to be expressed in 100% of epithelium examined, with expression levels ranging from low to high intensity, but not relating to the development or progression of the cancer. Cav-1 expression was not seen in normal and BPH epithelium. In PCa, only 24 % were positive. There was a significant increase in the number of Cav-1 positive tissues (p<0.05) between Gleason scores 6 and 7, correlating positively with increasing Gleason score, but no other significant differences were detected. Small samples numbers hampered the statistical analyses in Gleason grades of 4/5 and 8-10. Most notably, Cav-1 expression was inconsistent within an individual prostate sample, with different cancer foci having vastly different Cav-1 expression levels, suggesting different primary cancer phenotypes are present within a single patient. This raises the question of whether one cancer focus is more likely to progress to a more aggressive cancer type than its neighbour. In Chapter 5, the progression of PCa to a castrate resistant phenotype non-responsive to treatment was investigated, with particular reference to cholesterol metabolism and steroidogenesis. Cholesterol manipulation during reporter assay experiments from Chapter 3 raised questions regarding the role of this molecule in PCa cells. The effect that cholesterol elicits upon AR may be via its role in canonical signalling transduction pathways, such as those that are caveolae-mediated, or it may have a more direct role of manipulating AR activity. Results demonstrating de novo androgen synthesis in PCa cells suggests these cells have steroidogenic properties enabling survival in androgen-depleted environments. A well-defined steroidogenic pathway, whereby cholesterol is converted to testosterone, is facilitated by the enzymes CYP11A1, CYP17A1, 17β3HSD and 3β2HSD. Protein and mRNA for these enzymes were analysed in the six prostate-derived cell lines introduced in Chapter 3. mRNA results confirmed all six cell lines express CYP11A1 and 17β3HSD while 3β2HSD and CYP17A1 were expressed in two cell lines and one cell line respectively. The steroidogenic potential of the PCa cell lines LNCaP (androgen-dependent) and 22Rv1 (androgen-independent but responsive) was also compared. Charcoal-stripped steroid-negative medium, charcoal-stripped medium plus 10 nM DHT and charcoal-stripped medium plus 125 mM cholesterol were studied in cell culture. Appropriate controls were used. Expression of the steroidogenic proteins were analysed by Western blot analysis. Confocal microscopy was used to confirm mitochondrial localisation of CYP11A1 in LNCaP cells. Liquid chromatography-mass spectrometry analyses following ethyl acetate extractions of cell lysate and conditioned growth media demonstrated LNCaP and 22Rv1 cells synthesized testosterone in vitro. These results suggest that PCa cells have steroidogenic potential and may be able to drive their own growth even during ADT. In Chapter 6, expression and localisation of steroidogenesis enzymes, selected from Chapter 5 and known to convert cholesterol to testosterone, were examined by immunohistochemistry using the set of samples mentioned in Chapter 4. The enzymes selected were CYP11A1, CYP17A1, 17β3HSD and 3β2HSD. Detectable levels of protein for each enzyme in prostate tissue was as follows: CYP11A1 71 %, CYP17A1 28 %, 17β3HSD 82 % and 3β2HSD 19 %. All four enzymes were expressed simultaneously in approximately 10 % of the PCa cancer sections. CYP11A1 expression in glandular epithelium was found in 100 % of BPH samples (n=6). In PCa samples, 65 % of Gleason score 6 (n=23), 59 % of Gleason score 7 (n=27), and 100 % of Gleason score 8 (n=3) and 9 (n=4) expressed CYP11A1. CYP11A1 expression in BPH suggests prostate tissue has steroidogenic potential before PCa develops. Expression patterns varied amongst the samples, with some cells positive and some cells negative in most individual samples. Overall, only four samples, less than 6 %, were negative for all four steroidogenic enzymes. It is possible that ADT selects for CYP11A1-expressing cells amongst the heterogenic cell population of PCa and BPH, thereby contributing to cancer progression or recurrence. In summary (Chapter 7), prostate cancers retain the ability to respond to androgens in the initial stages of cancer development, but progressively become independent of exogenous androgens in advanced stages of the disease while maintaining expression of functional AR. The current study confirmed that AR has an inter-relationship with Cav-1 and their interaction within caveolae has a positive effect upon AR activity. It is generally accepted that the AR and Cav-1 relationship relies upon caveolae formation and the interaction of Cav-1 with cholesterol. Experiments involving cholesterol manipulation prompted us to the hypothesis of a more integral role between this molecule and AR. We investigated four classical steroidogenic enzymes, in prostate cell lines and tissue that are capable of converting cholesterol to testosterone, and the results suggest that prostate cancers may possess steroidogenic potential. Thus, when advanced PCa is treated with ADT, the therapy may be providing a micro-environmental selection pressure that favours PCa cells expressing steroidogenic enzymes. This ultimately drives them toward androgen self-sufficiency. The results may lead to a better understanding of phenotypic changes involving AR, Cav-1 and cholesterol that occur in PCa during its progression towards CRPC. Variable expression profiles of the proteins investigated within PCa tissues suggests that subtle differences in PCa phenotypes exist, not only between patients, but also within an individual. This fact highlights the complexity of this disease and presents a reason why current therapeutics fail. Future consideration must be given to the multi-focal nature of PCa, and the high probability that ADT nurtures therapy-resistant disease, to ensure development of more appropriate and individualised therapies.
Keyword prostate
androgen receptor
Additional Notes 35,43,48,57,60,64,67,70,89,97,124,153,154,156,159,160,163,164,166,167,175,177,182,184,187,191,204,206,207,209,210,212,214,216,217,219,220,265

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Created: Mon, 13 Sep 2010, 16:15:32 EST by Mr Nigel Bennett on behalf of Library - Information Access Service