Prostate cancer is a leading cause of cancer-related death in men, and while many men diagnosed with the disease will have an indolent clinical course, 20-25% of men will experience disease recurrence which is invariably lethal. There is an urgent need for prognostic biomarkers that will predict disease recurrence and risk-stratify patients upon diagnosis, allowing for personalised therapies. This thesis attempts to identify new prognostic biomarkers for prostate cancer and investigates their patterns of protein expression in human primary prostate tumour tissue. Cancer stem cells are cancer cells thought to be uniquely capable of self-renewal and tumorigenicity, and may have a role in tumour recurrence. Using a literature searching approach, potential biomarkers related to stem cells, cancer stem cells or recurrence in prostate cancer were identified, and ALDH7A1, BMI1, SDC1, MUC1-C, Nestin and ZSCAN4 were chosen for investigation. An in silico approach was also used for biomarker identification, with RS1 and SLC31A1 selected as their mRNA was found to be upregulated in recurrent tumours. The expression patterns of all 7 potential biomarkers were examined by immunohistochemistry on prostate tumour tissue and benign tissue from prostate biopsies and prostatectomies. BMI1, ALDH7A1, MUC1-C and Nestin showed no relationship to recurrence or other clinical features. RS1 protein levels increased in patients with recurrence within 5 years, negatively correlated with AR expression, and a meta-analysis showed that the RS1 gene was amplified in up to 32% of castration-resistant prostate tumours. ZSCAN4 was heterogeneously expressed in a subset of 26% of prostate tumours with unclear characteristics and was not expressed in benign tissue, but was not associated with recurrence. Finally, SDC1 expression was lost in tumour epithelium, but a population of unidentified SDC1-expressing cells were found in the stroma of a third of tumours, and an increased burden of these cells was associated with primary Gleason pattern 5 tumours. These cells do not overlap with common epithelial, mesenchymal or stromal lineages, but may be migratory. In summary, the data presented in this thesis identifies 3 potential new biomarkers for prostate cancer, and provides the basis for future characterisation of their wider roles in the disease.

<p>There is increasing evidence that prostate tumours are organized as a hierarchy with rare cancer stem cells (CSCs) implicated in initiating and maintaining the tumour. However, prospective prostate cancer stem cells (PCSCs) have not been thoroughly characterized from primary tissue specimens. Using the DU145 cell line, PCSCs have been propagated as non-adherent spheres <em>in vitro</em>. Approximately 1.25% of monolayer DU145 cells formed primary spheres while 26% of sphere cells formed subsequent spheres; a measure of PCSC self-renewal capacity. Spheres are enriched for cells expressing prostate basal and luminal cytokeratins and CSC markers (CD44, CD24, integrin alpha2beta1). PCSCs initiate xenograft tumours with enhanced capacity compared to monolayer cells. While epidermal growth factor (EGF) promoted PCSC propagation, basic fibroblast growth factor (bFGF) inhibited these events. Activation of EGF receptor (EGFR) signalling, following EGF treatment or expression of constitutively-active EGFR (EGFRvIII), increased sphere formation. Conversely, attenuation of EGFR signalling inhibited PCSC self-renewal. Consistent with the MEK-ERK pathway being a major target of EGFR signalling, the MEK-ERK pathway contributes to EGFR-facilitated PCSC propagation. Inhibition of ERK activation following MEK inhibitor treatment, expression of dominant-negative MEK1(K97M), or knockdown of ERK1 or ERK2 reduced PCSC propagation. Therefore, EGFR signalling promotes PCSC self-renewal by activating the MEK-ERK pathway.</p> <p>SOX2 is an essential transcription factor for stem cells, however, its role in PCSCs remains unclear. SOX2 protein is upregulated in PCSCs propagated as spheres, and its expression is regulated by EGFR signalling. EGFR activation, following EGF treatment or expression of constitutively-active EGFRvIII, increased SOX2 expression and PCSC self-renewal, while being attenuated by EGFR inhibitor treatment. Ectopic SOX2 expression enhanced EGF-induced PCSC self-renewal, while SOX2 knockdown renders PCSCs non-responsive to EGF-induced self-renewal and reduced their anchorage-independent growth. Furthermore, SOX2 expression is associated with the ability of PCSCs to form aggressive xenograft tumours. Collectively, SOX2 regulates EGFR-mediated PCSC self-renewal.</p> / Doctor of Philosophy (PhD)