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Type: Thesis
Title: Androgen signalling in the prostate cancer microenvironment.
Author: Leach, Damien Alexander
Issue Date: 2014
School/Discipline: School of Medicine
Abstract: Prostate cancer remains the second leading cause of cancer related death in Australian males, with approximately 28000 Australian men being diagnosed in 2007 and more than 3000 deaths as a result of this disease. The probability of patient survival from prostate cancer is greatly diminished when the disease has spread outside of the confines of the prostate. Disease spread, or the potential for spread, also determines the therapy received by the patient, be it surgical removal, chemotherapy, radiotherapy, or hormonal therapy, or any of these in combination. The advent of serum PSA testing has allowed for both earlier and increased detection of prostate cancer over the past 20 years. Despite this, the mortality rate for prostate cancer has remained relatively constant. Moreover, it is thought that increased detection may lead to over diagnosis and over treatment of indolent disease in up to 50% of cases, burdening patients who would not actually die from prostate cancer. Central to this issue is that there is no accurate means of predicting, at the time of diagnosis, the likelihood of prostate cancer becoming aggressive and metastasising, and thus identifying the patient population that would benefit most from more aggressive treatment. The prostate is a glandular structure composed of secretory epithelial cells embedded in a stroma containing smooth muscle cells, fibroblasts, and extracellular matrix (ECM) proteins, as well as immune cells and vasculature. Prostate cancer arises from the epithelial cells, which can subsequently evolve the capacity to breach the basement membrane, navigate through the surrounding stroma and ultimately enter the bloodstream allowing metastasis to distant sites such as bone. Recent studies suggest that each of these processes depends on a bi-directional interaction between the cancer cells and the prostate microenvironment, and moreover that structural or functional changes in that microenvironment are a necessary component of progressive tumourigenesis. As an example, activated cancer associated fibroblasts (CAFs) are thought to secrete paracrine factors that provide a more supportive environment, increasing cancer cell initiation and subsequent tumour growth. Another key aspect of prostate cancer development and maintenance are androgens, which act through their cognate receptor, the androgen receptor (AR). Current therapy for prostate cancer includes androgen deprivation therapy (ADT), which aims to ablate AR action. This therapy is most commonly used for advanced metastatic disease, however, in the USA between 1998 and 2005, ADT alone or in conjunction with radiotherapy was used as a primary treatment for advanced but organ confined prostate cancer in 70% of men. Studies have since shown an increased likelihood of disease progression and re-emergence in such cases. An explanation for the increased risk of progression may reside in understanding the role of AR in CAFs. Previously, CAF AR has been shown to be required for cancer initiation and tumour development. However, the specific cellular and molecular effect of androgens on fibroblasts is not well known, and the role of fibroblast AR in mediating progressive tumourigenesis of the cancer epithelia is unclear. The AR is a transcription factor which, upon activation by cellular internalized androgen, binds to DNA and mediates biological functions such as proliferation. Our understanding of the molecular actions of the AR are, however, almost entirely based on studies of its action in isolated prostate cancer epithelial cells, and inferences from the studies of other members of the nuclear receptor superfamily of ligand activated transcription factors. In general terms, the AR mediates the biological actions of androgens by becoming activated, translocating to the nucleus, and acting in concert with accessory proteins and coregulators to control chromatin structure and transcription at specific genomic sites. Whether these same general principles apply in fibroblasts is not known. In this thesis, human prostatic myofibroblast cell lines expressing AR (PShTert-AR) were compared to AR negative fibroblasts (PShTert-ctrl) and epithelial cancer cell lines (C4-2B), with view to defining the molecular and physiological roles of fibroblast AR. Microarray profiling revealed that only 10% of genes regulated by androgens in fibroblasts were also regulated in epithelial cells. Genes regulated by AR in fibroblasts were involved in pathways such as proliferation, ECM production, and adhesion. Conversely, in epithelial cells, AR regulated genes were involved in apoptosis, mitosis, and signal transduction. Functional studies showed that AR has an intrinsic anti-proliferative action in fibroblasts, but also promotes the release of paracrine factors that enhance growth of prostate cancer cells, both in vitro and in vivo. Androgens also induced fibroblast-mediated deposition of ECM proteins, such as collagen, whilst reducing the expression of enzymes involved in ECM degradation such MMP1 and MMP3. Further in vitro testing, demonstrated that ECM produced under androgenic stimulation increased attachment of the fibroblasts themselves by 60% in under 30 minutes (p<0.05), as well as attachment of prostate cancer epithelial cells by up to 35% (p<0.05). In addition, androgens increased fibroblast cell adhesion and suppressed motility and invasion over a four hour period by 30% (p<0.05). Utilizing several sets of microarray data, qualitative and quantitative differences in expression of AR coregulators in fibroblasts and epithelial cells were also determined. Intriguingly, it was observed that a number of previously described epithelial-cell AR coregulators were expressed almost exclusively in fibroblasts, including several proteins such as the coactivator Hic-5 (TGFB1I1), known to interact with focal adhesions. Despite evidence in previous studies for expression predominantly in fibroblasts, Hic-5 has been investigated almost exclusively in a small number of epithelial cell lines. Here, the biological roles of this AR coregulator were investigated using isogenic myofibroblasts with or without Hic-5, and in a transient siRNA model of Hic-5 depletion. Microarray and RT-qPCR investigation revealed that the level of Hic-5 affects the expression of over 50% of androgen mediated genes, and in the majority of cases (85%) works to amplify (coactivate) the natural androgenic response. Supporting this finding, depletion of Hic-5 decreased the androgenic response from 34 of the 69 tested AR-responsive luciferase reporter constructs tested (p<0.01). Finally, in a novel and important finding, it was demonstrated that androgens result in a dynamic cellular localisation of Hic-5 in fibroblasts, and that this process is a key determinant of the observed androgen-mediated responses, adhesion and motility. Collectively, these data demonstrate that Hic-5 is an important modulator of both androgen mediated gene transcription and androgen driven cell movement in fibroblasts. Furthermore, these studies demonstrate that the cell specificity of AR signalling may be mediated by unique interactions with pools of coregulators specific to fibroblasts, and that changes in these pools could significantly alter androgen signalling within the cancer environment. In order to assess the physiological relevance of fibroblast AR, a tissue microarray was constructed as part of this thesis that contained both benign and malignant prostate samples from 64 patients, on which quantitative immunohistochemical assessment of AR expression was performed in both epithelial and stromal compartments. Changes in epithelial AR expression did not affect patient outcome, but were significantly related to Gleason grade (p<0.001) and serum PSA (p<0.001). In contrast, expression of AR in stroma were significantly associated with prostate cancer related death (p<0.05). This relationship was found to be unique to the cancer associated stroma, as AR expression in patient-matched benign tissue did not associate with any of the clinical parameters. Combined with the in vitro findings in this thesis, those data strongly suggest that although stromal AR is required for cancer initiation and growth, a subsequent loss of stromal AR produces a cancer more likely to be associated with a detrimental patient outcome. The findings from this thesis identify two mechanisms by which AR signalling in fibroblasts may contribute to prostate cancer progression. First, a loss of stromal AR results in increased fibroblast movement and proliferation, and the secretion of a permissive ECM for cancer cell detachment and movement. Second, AR activation by androgen results in redistribution of fibroblast-specific cofactors such as Hic-5 from the cytoplasm to the nucleus, leading to enhanced AR transcriptional activity. The movement of Hic-5 from focal adhesion sites concomitantly increases adhesion and reduces motility. By increasing the understanding of the molecular pathways and function of AR and androgens in the stroma, the research undertaken in this thesis has been able to establish new models of AR action in the cancerous environment. Significantly, these data provide a mechanism for the increased likelihood of disease progression and re-emergence upon treatment of advanced organ confined prostate cancer with ADT, as a loss of fibroblast AR activity and altered ECM production is predicted to enable cancer metastasis. This thesis concludes that dysregulation of AR action in the stroma potentiates prostate cancer progression, and may thus be used as an indicator of cancer aggressiveness and metastasis.
Advisor: Buchanan, Grant
Need, Eleanor Frances
Trotta, Andrew Paul
Marshall, Villis Raymond
Dissertation Note: Thesis (Ph.D.) -- University of Adelaide, School of Medicine, 2014
Keywords: prostate cancer; stroma; fibroblasts; androgen receptor
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