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Gene Expression Patterns in Human Prostate Stem Cell Differentiation

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Gene Expression Patterns in Human Prostate Stem Cell Differentiation Gene Expression Patterns in Human Prostate Stem Cell Differentiation Emma Elizabeth Oldridge PhD University of York Department of Biology September 2012 ABSTRACT The identification of phenotypic differences between stem cells (SCs) and their more differentiated counterparts is crucial for designing novel SC-based therapeutics for prostate cancer (CaP). RARRES1 and LXN were identified as two homologous genes whose expression was highly significantly down-regulated in the SC fraction compared to more differentiated epithelial cells. The overall aim of this study was to investigate the expression, regulation and function of the SC-silenced genes RARRES1 and LXN, and their potential interacting partner CPA4 in prostate epithelial differentiation and CaP. We showed that RARRES1 and LXN were SC-silenced genes, whose expression was induced by the pro-differentiation agent all-trans retinoic acid (atRA). AtRA induced expression to a higher extent in the most differentiated cells than the SC fraction, suggesting that this sub- population was less responsive to atRA. Importantly, siRNA suppression of RARRES1 and LXN enhanced the SC properties of primary prostate cultures, as shown by a significant increase in their colony forming ability. Expression of both RARRES1 and LXN was co-ordinately repressed by DNA methylation in CaP cell lines and inhibition of RARRES1 and LXN increased the invasive capacity of primary prostate cultures, which also fully rescued an inhibitory effect induced by atRA. Despite their homology and adjacent location on chromosome 3, we provide evidence that RARRES1 and LXN reside within different sub-cellular compartments; RARRES1 is not a plasma membrane protein as previously supposed but is located in the endoplasmic reticulum, while LXN is localised to the nucleus of prostate epithelial cells. These data provide novel results identifying two potential tumour suppressor genes as co- ordinately regulated, SC-silenced genes that function to suppress the invasion and colony forming ability of CaP cells. Work now should be focussed on determining whether re- administration of RARRES1 and LXN would be a valid differentiation strategy for the treatment, and potentially eradication, of CaP. 2 LIST OF CONTENTS ABSTRACT ........................................................................................................ 2 LIST OF CONTENTS.......................................................................................... 3 LIST OF FIGURES ............................................................................................. 9 LIST OF TABLES ............................................................................................. 13 ACKNOWLEDGEMENTS................................................................................. 14 AUTHOR’S DECLARATION ............................................................................ 14 1. INTRODUCTION ...................................................................................... 17 1.1. Prostate anatomy ..............................................................................................17 1.1.1. Prostate development ................................................................................17 1.2. Disorders of the prostate ..................................................................................19 1.3. Cellular organisation of the prostate epithelium ..............................................21 1.3.1. Prostate epithelial stem cells ......................................................................23 1.3.2. Epithelial hierarchy within the prostate epithelium ......................................26 1.4. Prostate cancer .................................................................................................28 1.4.1. Cancer stem cells ......................................................................................28 1.4.2. Prostate cancer stem cells .........................................................................30 1.4.3. Epidemiology of prostate cancer ................................................................31 1.4.4. Diagnosis of prostate cancer ......................................................................32 1.4.5. Current treatments for prostate cancer .......................................................36 1.4.5.1. Low grade organ-confined prostate cancer ..................................36 1.4.5.2. High grade metastatic prostate cancer ........................................37 1.4.5.3. Differentiation therapy as an alternative treatment for prostate cancer ........................................................................................38 1.4.6. Castration-resistant prostate cancer ...........................................................39 1.4.7. Cancer cell invasion and metastasis ..........................................................41 1.4.8. Epithelial-to-mesenchymal transition ..........................................................44 1.5. Regulation of gene expression in the prostate epithelium ..............................45 1.5.1. Retinoic acid signalling ..............................................................................46 1.5.2. Genetic changes in cancer .........................................................................47 3 1.6. Epigenetic regulation mechanisms ..................................................................50 1.6.1. DNA methylation ........................................................................................50 1.6.2. Histone modifications and chromatin remodelling .......................................53 1.6.3. MicroRNAs ................................................................................................55 1.6.4. Epigenetic therapy in cancer ......................................................................55 1.6.4.1. DNA demethylating agents..........................................................55 1.6.4.2. HDAC inhibitors ..........................................................................56 1.6.5. Epigenetic changes in prostate cancer .......................................................57 1.6.5.1. DNA methylation .........................................................................57 1.6.5.2. Chromatin Remodelling ...............................................................59 1.6.5.3. MicroRNAs .................................................................................59 1.6.6. Epigenetic changes in stem cells ...............................................................60 1.7. Identification of a prostate cancer stem cell signature ....................................62 1.7.1. Retinoic acid receptor responder 1 (RARRES1) .........................................62 1.7.2. Latexin (LXN) ............................................................................................64 1.7.3. Carboxypeptidase A4 (CPA4) ....................................................................66 1.8. Prostate models.................................................................................................67 1.8.1. Prostate epithelial cell lines ........................................................................67 1.8.2. Primary prostate epithelial cultures ............................................................67 1.8.3. In vivo mouse models ................................................................................69 1.9. Aims of research ...............................................................................................70 2. MATERIALS AND METHODS ................................................................. 71 2.1. Mammalian cell culture .....................................................................................71 2.1.1. Maintenance of mammalian cell lines .........................................................71 2.1.2. Isolation and maintenance of primary epithelial cultures .............................71 2.1.3. Generation and maintenance of xenografts ................................................72 2.1.4. Embedding and sectioning of snap-frozen xenograft tissue ........................72 2.1.5. Cryopreservation of mammalian cells .........................................................74 2.1.6. Irradiation of fibroblasts ..............................................................................74 2.1.7. Fetal calf serum hormone depletion ...........................................................74 2.1.8. Determination of live cell number using a haemocytometer ........................74 2.1.9. Determination of viable cell number using the Vi-Cell cell viability analyser 74 2.2. Primary culture enrichment ..............................................................................75 4 2.2.1. Enrichment of α2β1-integrin expressing cells ...............................................75 2.2.2. Enrichment of CD133 expressing cells .......................................................75 2.3. Drug treatment of cells ......................................................................................76 2.3.1. Treatment of cell lines with NaBu, TSA and 5-Aza-dC ................................76 2.3.2. Treatment of cell lines and primary cultures with atRA................................76 2.4. Luciferase assay................................................................................................76 2.5. Transfection of mammalian cells ......................................................................77 2.5.1. Transfection of cell lines with siRNA...........................................................77
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