Delineating a Functional Role for the Urinary Biomarker Lipocalin 2 in Prostate Cancer
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Prostate cancer (PCa) is the most commonly diagnosed cancer amongst Western males. PCa progression is strongly linked to steroid receptor signalling, however the modulation of steroid receptor expression in PCa is incompletely understood. Lipocalin 2 (LCN2) is a secreted protein which binds to Fe3+-containing siderophores and was originally identified as part of the innate immune response. LCN2 has been proposed as a potential biomarker for a range of cancers. However, LCN2 effects appear to be tissue specific. LCN2 expression is associated with poor prognosis in breast cancer, but with good prognosis in pancreatic cancer where it has been used therapeutically. The role of LCN2 in prostate cancer is poorly understood, in particular its effects on steroid receptor regulation. To elucidate the role of LCN2 in prostate cancer, the LCN2 gene was ectopically expressed in LNCaP cells to generate the LNCaP-LCN2 cell line. LNCaP-LCN2 cells had elevated androgen receptor expression which was linked to increased levels of KLK3 (PSA). LNCaP-LCN2 cells also had reduced levels of Estrogen receptor α (ERα), but increased expression of ERβ. This was combined with higher levels of E-cadherin, but not to changes in other EMT markers. Reciprocally, LCN2 was suppressed using RNAi in the PC3 cell line to generate PC3-shLCN2 cells. PC3-shLCN2 displayed a distinct change in morphology, with increased cell size and a sub-population of multi-nucleated and highly enlarged cells. PC3-shLCN2 cells had reduced proliferation, and lost the ability to form colonies in a 3D substrate. With regards to steroid receptors, PC3-shLCN2 cells had increased ERα expression, but reduced ERβ expression. This was also combined with a loss of E-cadherin and EGFR. Microarray analysis of PC3-shLCN2 cells identified changes to expression of a wide range of genes including VEGF-R, SPARC and KLK6. Functional grouping of differentially expressed genes suggests that LCN2 in involved in a range of cellular processes including hormone receptor response, Wnt signalling and cell cytoskeletal integrity. Many, but not all genes identified by microarray were responsive to recombinant LCN2 protein indicating a paracrine function for the protein. Treatment of PC3 cells with the iron chelator Deferoxamine resulted in phenotypic changes similar to those found in PC3-shLCN2 cells which suggest that LCN2 functions in part due to intracellular iron regulation. In summary, the data presented in this thesis suggests that LCN2 has both pro- and anti- tumourigenic properties in prostate cancer and that the protein is involved in a much wider range of functions than previously described.
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