ANDROGEN RECEPTOR, CAVEOLIN-1 AND ANDROGEN SELF-SUFFICIENCY IN PROSTATE CANCER

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 (