Src signaling plays an important role in prostate cancer (PrCa) progression. It has previously been shown that Src interacts with androgen receptor (AR) and enhances AR transactivation. Although it has been shown that Src promotes AR activity, the underlying pathway has not been defined. To help characterize the Src-AR pathway, the cellular localizations of Src, p-Src, AR, pAR, and Prostate Specific Antigen (PSA, an AR target gene) were analyzed in androgen-dependent (AD) LNCaP cells and in androgen-independent (AI) castration-resistant C4-2B cells. Using sub-cellular fractionation, the data showed that treatment of AD cells with synthetic androgen R1881 increased p-Src, AR, pAR, and PSA in the nucleus, while the levels of c-Src remained unchanged. Treatment of AI cells with R1881 increased pSrc and AR in the nucleus, while the levels of c-Src and PSA remained unchanged. When using immunofluorescence microscopy, R1881 did not appear to increase the nuclear levels of p-Src or c-Src, so perhaps this technique is not as sensitive or quantitative as subcellular fractionation immunoblots. The presence of PSA in the nucleus was unexpected given its well proven role as a secreted protein. Nuclear PSA was observed upon androgen stimulation in AD and AI cells, and in the nucleus of AI cells upon androgen deprivation. Given PSA's ability to induce cell division and decrease apoptosis when transfected into cells, its presence in the nucleus may imply that PSA acts there to help induce tumorigenesis. The effect of Src on AR activity was further studied by transfection of a dominant negative src (SrcK298M) in AD and AI cells. Transfection with SrcK298M did not affect PSA expression in LNCaP cells, but strongly inhibited PSA levels in AI cells. Integrin signaling through Src was investigated in PrCa by ligand binding assay in AD and AI cells. The data showed that alpha v beta 3 integrin (but not alpha v beta 6) upon attachment to fibronectin or TGF-beta-latency associated peptide (TGF- beta-LAP) increases p-Src levels in AD and AI cells, while the levels of c-Src, PSA, and AKT remain unchanged. Thus, alpha v beta 3 integrin facilitates Src signaling, but the activation does not appear to affect AR transactivation. In conclusion, these data show that Src is required for AR activity and, consequently, PSA expression in AI prostate cancer cells, but not in AD cells. These data also suggest that the nuclear co-localization of p-Src, AR and PSA might allow macromolecular interactions, which can further enhance AR transactivation and promote disease progression. With respect to the switch in tumor progression from an AD to AI state, the data indicate that the integrin-Src pathway does not include AKT or PSA (and not AR by deduction), so perhaps other non-AR pathways help facilitate tumor growth at the AI state.

Background: The first-line treatment for patients with advanced prostate cancer (PC) is androgen deprivation therapy. This therapy is initially effective, but after some time tumors relapse, predominantly within the bone, and are then termed castration-resistant prostate cancer (CRPC). The majority of CRPC tumors show androgen receptor (AR) activity despite castrate levels of circulating testosterone. AR activity could be caused by several mechanisms including; intratumoral androgen synthesis, AR amplification, AR mutations and expression of AR splice variants. The mechanisms controlling CRPC growth in the clinically most relevant metastatic site, the bone, are not fully identified. The purpose of this thesis was therefore to explore AR expression and possible mechanisms behind CRPC growth in PC bone metastases in order to find mechanisms that could be targeted for treatment and/or predict response to certain therapies. Materials and Methods: We have examined hormone-naïve and CRPC bone metastases samples obtained from patients at metastasis surgery, non-malignant and malignant prostate samples obtained from patients at radical prostatectomy, and PC cell lines cultured in vitro. Analysis has been performed using RT-PCR, whole-genome expression arrays, immunohistochemistry, western blotting, FISH, copy number assays and gene ontology analysis. Functional studies have been made by protein overexpression and knock-down in PC cells in vitro and effects studied by evaluation of cell viability, migration, and invasion. Results: We found that high nuclear AR immunostaining (presumed to reflect high AR activity) in bone metastases from CRPC patients was associated with a particularly poor prognosis, while no difference in AR staining was observed between hormone-naïve and CRPC metastases. Further, expression of AR splice variants (AR-V7, AR-V567es) was associated with a high nuclear AR immunostaining score and shown to be increased in CRPC compared to hormone-naïve bone metastases. High levels (levels in the upper quartile) of AR splice variants in CRPC bone metastases was related to disturbed cell cycle regulation and short patients survival. No differences in steroidogenic enzyme levels were detected between CRPC and hormone-naïve bone metastases. Higher levels of enzymes involved in late steps of androgen synthesis (adrenal gland steroid conversion) were observed in bone metastases than in non-malignant and/or malignant prostate tissue, while the enzyme levels in earlier steps (de novo steroidogenesis) were lower in bone metastases. A subgroup of metastases expressed very high levels of AKR1C3, indicating that this group may have an induced capacity of converting adrenal-gland derived steroids into more potent androgens. This was not associated to CRPC but merely with the advanced stage of metastasis. High protein levels of AR splice variants were found in bone metastases with low AKR1C3 levels, while metastases with high AKR1C3 levels primarily contained low AR variant levels. Furthermore, about half of the CRPC bone metastases showed androgen receptor gene amplification which was associated with co-amplification of YIPF6, and a gene expression pattern that pointed at decreased osteoclast activity, and consequently decreased bone resorption. Conclusions: The majority of CRPC bone metastases show high nuclear AR immunostaining that seems to be associated with a particularly unfavorable outcome after metastasis surgery. Subgroups of CRPC bone metastases could be identified according to presence of AR amplification and expression levels of AKR1C3 or AR splice variants, which might have clinical relevance for treatment of PC patients. / <p>Författaren är även publicerad med efternamnet Hörnberg.</p>

Prostate cancer is the second leading cause of death from cancer in Australian men. As prostate cancer cells are reliant on androgens for growth and survival, the standard therapy for metastatic disease is androgen ablation therapy (AAT). AAT inhibits androgen signalling by altering androgen synthesis or prevent binding of androgens to their intracellular mediator, the androgen receptor (AR). Although initially effective, virtually all patients relapse, beyond which there are limited treatment options. The failure of AAT is not necessarily due to a decreased requirement for androgen signalling, but rather the AR is able to maintain signalling and tumour growth in an androgen-depleted environment. Therefore novel strategies that directly target the AR may provide a more effective therapeutic approach. We have endeavoured to suppress AR activity in prostate cancer cells by utilising a dominant negative AR. The most effective dominant negative construct developed, ARi41O, lacks amino acids 39-410 in the AR amino terminal transactivation domain. In studies of transcriptional activity, ARi410 has no intrinsic activity but inhibits the activity of wild type AR (wtAR) and also clinically relevant AR variants, by up to 95%. The objective of this thesis was to characterise the mechanisms of action of ARi410 and assess the functional effects of introducing this dominant negative receptor into prostate cancer cells. To investigate the mechanism by which ARi410 suppresses AR activity, a robust and sensitive AR inhibition assay was developed. This assay revealed that ARi410 is a potent inhibitor of AR activity on three independent AR-regulated promoters, regardless of the level of AR expression. Furthermore, while ARi410 can inhibit AR activity, it does not alter AR protein levels. By using ARi410 variants with mutations and/or deletions in regions of functional importance, the AR inhibition assay was also used to identify the critical regions of ARi410 required for its dominant negative activity. These studies demonstrate that the dominant negative activity of ARi41 0 is ligand-dependent, requires dimerisation through the ligand binding domain (LBD) and an intact DNA-binding domain (DBD). Further investigation into the mechanism of dominant negative activity revealed that ARi410 does not alter the subcellular localisation of AR, as both receptors are predominantly cytoplasmic in the absence of ligand and rapidly co-localise to the nucleus in response to androgens. Furthermore, an interaction between AR and ARi410 was observed in the presence and absence of ligand, and electrophoretic mobility shift assays demonstrated that AR and ARi410 form heterodimers on DNA. These studies led to the conclusion that the mechanism of dominant negative activity by ARi4I0 involves the formation of inactive receptor heterodimers that assemble on DNA and suppress AR activity. To determine the functional consequence of expressing the dominant negative androgen receptor in prostate cancer cells, an adenoviral method of gene delivery was developed. Adenoviral expression of ARi410 in LNCaP prostate cancer cells did not allow assessment of cell viability due to cell-specific toxicity of the viral vectors when expressed long-term. However, short-term expression of ARi410 in LNCaP cells resulted in inhibition of AR signalling, as determined by reduced expression of the androgen regulated genes apolipoprotein D and kallikrein 2. Importantly, this finding is consistent with the inhibitory activity of ARi410 observed using synthetic AR-regulated reporter genes in the AR inhibition assay, and demonstrates that ARi410 can effectively suppress endogenous AR signalling. The results of this thesis indicate that heterodimerisation between AR and ARi410 is the most likely mechanism of dominant negative inhibition of AR function by ARi410, and that the DBD and dimerisation through the LBD are required for optimal dominant negative activity. Furthermore, this thesis has demonstrated that ARi410 is an effective inhibitor of AR signalling and provides a basis for further functional studies and evaluation of the dominant negative androgen receptor in vitro and in vivo. / http://proxy.library.adelaide.edu.au/login?url= http://library.adelaide.edu.au/cgi-bin/Pwebrecon.cgi?BBID=1338478 / Thesis (Ph.D.) -- University of Adelaide, School of Molecular and Biomedical Science, 2008

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