High mobility group A2 (HMGA2) is a non-histone protein highly expressed during the development but is low or absent in most adult tissues. Epithelial-mesenchymal transition (EMT) plays a critical role in prostate cancer progression and metastasis. HMGA2 has been shown to promote EMT in separate studies. Interestingly, wild-type HMGA2 and truncated (lacking the 3’UTR) HMGA2 isoforms are overexpressed in many cancers. However, there are no studies on the role of each isoform in prostate cancer progression. We hypothesized that wild-type and truncated HMGA2 promotes prostate cancer progression by different mechanisms. We analyzed the expression of HMGA2 in the prostate panel by western blot analysis and the localization in prostate tissue microarray by immunohistochemistry. We stably overexpressed wild-type and truncated HMGA2 cDNA in LNCaP cells and measured the expression and the localization of HMGA2 as well as EMT markers. We also performed the migration and cell viability assays. We analyzed phospho-ERK in cells overexpressing HMGA2 as well as inhibition with U0126 (MAPK inhibitor). To explore the role of truncated HMGA2, we measured the reactive oxygen species (ROS) concentration by DCFDA dye, as well as analyzing Jun-D as a putative downstream effector of HMGA2. Additionally, we knocked down Jun-D and performed the migration and cell viability assays. We treated ARCaP-M mesenchymal cells with camalexin, a 3-thizol-2-yl-indole (a natural product, as a candidate to target HMGA2) in vitro and in vivo in nude mice. Our results showed an increase in nuclear HMGA2 expression with prostate cancer progression as compared to normal tissue. LNCaP cells overexpressing wild-type but not truncated HMGA2 displayed nuclear localization and induced EMT via the ERK1/2 pathway, and this effect could be reversed by treating the cells with U0126. Conversely, truncated HMGA2 displayed cytoplasmic expression and increased prostate cancer migration via increasing Jun-D expression and ROS; this could be antagonized by Jun-D knockdown. Finally, treating ARCaP-M aggressive prostate cancer cells with camalexin reduce its expression in vitro and in vivo. In conclusion, both wild-type and truncated HMGA2 induce prostate cancer progression by different mechanisms which may be targeted by camalexin.

Radiotherapy is the most widely used treatment for cancer, with 4 out of 10 cancer patients receiving radiotherapy as part of their treatment. The delineation of gross tumour volume (GTV) is crucial in the treatment of radiotherapy. An automatic contouring system would be beneficial in radiotherapy planning in order to generate objective, accurate and reproducible GTV contours. Image guided radiotherapy (IGRT) acquires patient images just before treatment delivery to allow any necessary positional correction. Consequently, real-time contouring system provides an opportunity to adopt radiotherapy on the treatment day. In this thesis, freely deformable models (FDM) and shape constrained deformable models (SCDMs) were used to automatically delineate the GTV for brain cancer and prostate cancer. Level set method (LSM) is a typical FDM which was used to contour glioma on brain MRI. A series of low level image segmentation methodologies are cascaded to form a case-wise fully automatic initialisation pipeline for the level set function. Dice similarity coefficients (DSCs) were used to evaluate the contours. Results shown a good agreement between clinical contours and LSM contours, in 93% of cases the DSCs was found to be between 60% and 80%. The second significant contribution is a novel development to the active shape model (ASM), a profile feature was selected from pre-computed texture features by minimising the Mahalanobis distance (MD) to obtain the most distinct feature for each landmark, instead of conventional image intensity. A new group-wise registration scheme was applied to solve the correspondence definition within the training data. This ASM model was used to delineated prostate GTV on CT. DSCs for this case was found between 0.75 and 0.91 with the mean DSC 0.81. The last contribution is a fully automatic active appearance model (AAM) which captures image appearance near the GTV boundary. The image appearance of inner GTV was discarded to spare the potential disruption caused by brachytherapy seeds or gold markers. This model outperforms conventional AAM at the prostate base and apex region by involving surround organs. The overall mean DSC for this case is 0.85.

O objetivo deste estudo foi o de medir in vivo as dimensões da próstata, pelo exame ultra-sonográfico, de cães de diferentes tamanhos, com idade entre 1 e 5 anos. Foram realizados exames em 47 cães, sem processos patológicos aparentes. Os pacientes foram divididos em dois grupos de acordo com a idade, Grupo 1: cães entre 12 e 30 meses e Grupo 2: cães entre 31 e 60 meses. Cada grupo foi subdividido em três subgrupos de acordo com o peso. Subgrupos 1 e 4 com animais até 10kg, Subgrupos 2 e 5 com animais entre 11 e 25kg e Subgrupos 3 e 6 com animais acima de 25kg sendo que os Subgrupos 1 a 3 pertencem ao Grupo 1 e os Subgrupos 4 a 6 pertencem ao Grupo 2. As imagens em corte longitudinal e transversal permitiram a observação da próstata, localizada caudalmente à bexiga urinária, de formato arredondado, de contornos regulares e definidos e de ecogenicidade maior que a do baço. Comprimento e altura foram mensurados no plano longitudinal, já a largura foi medida no plano transversal. O Subgrupo 1 apresentou medidas prostáticas de comprimento: 1,19 (±0,16cm); de altura: 1,17 (±0,2cm) e de largura: 1,41 (±0,24cm), valores menores que os obtidos no Subgrupo 4 que apresentou comprimento: 2,14 (±0,13cm); altura: 2,12 (±0,16cm) e largura de 2,59 (±0,21cm). O Subgrupo 2 apresentou valores prostáticos de comprimento: 2,17 (±0,11cm); de altura: 2,04 (±0,15cm) e de largura: 2,64 (±0,13cm), valores menores que os obtidos no Subgrupo 5 que apresentou comprimento: 2,81 (±0,44cm); altura 3,73: (±0,37cm) e largura de 3,29 (±0,38cm). O Subgrupo 3 apresentou valores prostáticos de comprimento 3,09 (±0,21cm); de altura: 2,93 (±0,21cm) e de largura: 3,63 (±0,19cm), valores menores que os obtidos no Subgrupo 6 que apresentou comprimento: 3,47 (±0,31cm); altura: 3,35 (±0,35cm) e largura de 4,03 (±0,28cm). A correlação foi forte entre os valores prostáticos determinados e a massa corpórea dos cães estudados. / The aim of this study was ultrasonographically assess the prostatic dimensions in dogs of different sizes, aging from 1 to 5 years-old. Forty-seven examinations were done, in dogs with no clinical diseases. The patients were divided into two groups according to their ages, such as: Group 1 - from 12 to 30 months-old and Group 2 ? from 31 to 60 months-old. Each group was divided into 3 other groups according to their weight. Subgroup 1 and 4: up to 10 Kg, subgroup 2 and 5: from 11 to 25 Kg and subgroups 3 and 6: over 25 Kg (subgroups 1 to 3 were inserted in Group 1 and subgroups 4 to 6 were inserted in Group 2). The sagittal and transverse planes on ultrasonographic examination provided the entire visualization of the prostate, which was round to normal-shaped, lies caudally to the urinary bladder, with smooth margins and hyperechoic to the spleen. Their length and height were measured on sagittal plane and the width was measured on transverse plane. Regarding the subgroup 1, the mean prostatic length was 1,19 cm (± 0,16 cm), the mean prostatic height was 1,17 cm (± 0,2 cm) and the mean prostatic width was 1,41 cm (± 0,24 cm), and these measurements showed to be shorter than the ones from subgroup 4, such as 2,14 cm (± 0,13) as for the mean length, 2,12 cm (± 0,16 cm) as for the mean height and finally 2,59 cm (± 0,21 cm) as for the mean width. Regarding the subgroup 2, the mean prostatic length was 2,17 cm (± 0,11), the mean height was 2,04 (± 0,15 cm) and the mean width was 2,64 cm (± 0,13), and these measurements showed to be shorter than the ones from subgroup 5, such as 2,81 cm (± 0,44) as for mean length, 3,73 cm (± 0,37 cm) as for the mean height and finally 3,29 cm (± 0,38 cm) as for the mean width. Regarding the subgroup 3, the mean prostatic length was 3,09 cm (± 0,21), the mean height was 2,93 (± 0,21 cm) and the mean width was 3,63 cm (± 0,19), and these measurements showed to be shorter than the ones from subgroup 6, such as 3,47 cm (± 0,31) as for mean length, 3,35 cm (± 0,35 cm) as for the mean height and finally 4,03 cm (± 0,28 cm) as for the mean width. There was a high correlation between the prostatic dimensions and the body mass of theses dogs.

PANDER/FAM3B (PANcreatic-DERived factor) é uma citocina capaz de induzir apoptose em células-β secretoras de insulina e regular a homeostase da glicose nos tecidos periféricos. Considerando que PANDER/FAM3B também é expresso em tumores de próstata, o presente trabalho avaliou o papel desta citocina na inibição da apoptose in vitro, assim como o crescimento e invasividade tumoral de células de carcinoma de próstata da linhagem DU145 in vivo, usando o modelo de superexpressão estável mediada por lentivirus. Os nossos resultados apontam um aumento da viabilidade e uma diminuição da morte celular em células que superexpressam PANDER quando comparadas ao grupo controle. Este efeito protetor é acompanhado por um aumento da expressão de genes anti-apoptoticos e uma diminuição da atividade proteolítica das caspases. Por outro lado, a superexpressão de PANDER/FAM3B por tumores in vivo se correlaciona com aumento da massa tumoral e o aumento de vasos sanguíneos nos tumores. Em síntese, nossos dados demonstram que, em contraste ao papel observado em células β-pancreáticas, o PANDER/FAM3B inibe morte celular e promove a tumorigenicidade e o crescimento tumoral in vivo, sugerindo ao mesmo tempo, algum envolvimento com angiogênese e metástase em células DU145. / PANDER / FAM3B (pancreatic-derived factor) is a cytokine capable of inducing apoptosis in secreting β-cells and insulin to regulate glucose homeostasis in peripheral tissues. Whereas PANDER / FAM3B is also expressed in prostate tumors, this study evaluated the role of this cytokine in the inhibition of apoptosis in vitro as well as tumor growth and invasiveness of prostate carcinoma DU145 cells lineage in vivo, using the model Stable overexpression mediated by lentivirus. Our results suggest that increased viability and decreased cell death in cells that overexpress PANDER when compared to the control group. This protective effect is accompanied by an increased expression of anti-apoptotic genes and a decrease in proteolytic activity of caspases. Moreover, overexpression of PANDER / FAM3B by tumors in vivo correlates with increased tumor mass and the increase of blood vessels in tumors. In summary, our data demonstrate that in contrast to the role observed in pancreatic cells, PANDER / FAM3B inhibits cell death and promotes tumorigenicity and tumor growth in vivo, suggesting at the same time, some involvement in angiogenesis and metastasis in cell DU145.

No mundo inteiro, o carcinoma de próstata ocupa o quinto lugar entre as neoplasias malignas de maior mortalidade. No Brasil, estima-se para o ano de 2001 que, entre os tumores malignos no sexo masculino, o carcinoma de próstata terá a segunda maior taxa de mortalidade e a primeira taxa de incidência (Estimativa da incidência e mortalidade por câncer no Brasil – 2001 – INCA). Uma vez que a taxa de mortalidade por carcinoma de próstata na população brasileira tem aumentado significativamente nos últimos anos, a presente tese se propôs a investigar regiões polimórficas em genes conhecidos que poderiam estar associadas a um aumento na predisposição a esta forma de câncer. Assim sendo, estudamos as regiões polimórficas CAG e GGC do gene do receptor de andrógeno; o polimorfismo C1171T do gene do receptor de vitamina D; o polimorfismo D104N do gene da endostatina; o polimorfismo Pro72Arg do gene p53 e a região polimórfica AAAAC localizada na região 3’ não traduzida do gene MXI1. / In the world’s population prostate carcinoma is the fifth most commom male cancer-related death malignancy. In Brazil, among all male invasive cancers it is expected that prostate carcinoma will have the second highest death rate and the highest incidence rate (Estimativa da incidência e mortalidade por câncer no Brasil, 2001). As the prostate carcinoma death rate in brazilian population has been increasing over the last several years we proposed to investigate polymorphic regions of known genes that might be associated with prostate carcinoma predisposition. We studied the androgen receptor CAG and GGC polymorphic regions, the vitamin D receptor C1171T polymorphism, the endostatin D104N polymorphism, the p53 Pro72Arg polymorphism and the MXI1 AAAAC polymorphic region.

O câncer de próstata é o tumor maligno mais freqüente nos homens com idade superior a 50 anos, excetuando-se os tumores cutâneos. No Brasil estima-se para o ano de 2012 cerca de 60.180 casos novos deste tipo de neoplasia. Os marcadores tumorais permitem fazer o rastreamento do câncer, o diagnóstico diferencial entre uma neoplasia benigna e maligna, a avaliação de prognóstico e o acompanhamento terapêutico, assim como a detecção da recidiva tumoral. Dentre estes marcadores tumorais, tem-se dado muito atenção para as proteínas que mediam e participam da progressão do ciclo celular. A ciclina D1 é uma proteína nuclear de vida curta que é destruída pela via da ubiquitina ATP dependente, e está envolvida na transição celular da fase do ciclo G1 (repouso) para a fase S (síntese) tanto em células normais como em células neoplásicas. A super expressão de ciclina D1 remove a regulação normal do ciclo celular causando proliferação celular descontrolada, um crescimento anormal dos tecidos e a transformação para um fenótipo neoplásico, atuando como oncogene. No presente trabalho foi estudado a expressão de ciclina D1 em adenocarcinomas de próstata, tendo como objetivo avaliar a relação desta proteína com parâmetros epidemiológicos, clínicos e histopatológicos. Adicionalmente também foi feita comparação de escore de Gleason e lateralidade tumoral entre biópsias prostáticas com agulha e de prostatectomias radicais. No ensaio para ciclina D1 foram analisados 85 casos através de imunoistoquímica (IHQ) de material proveniente de prostatectomias radicais diagnosticados com adenocarcinoma de próstata entre os anos de 2005 e 2010 em nosso serviço. O método de avaliação se utilizou de microscopia ótica comum e contagem semi-quantitativa, comparado-se a expressão com achados clínicos, epidemiológicos e histopatológicos utilizando-se Teste T de Fisher, Qui Quadrado, Mann-Whitney, Curva ROC e correlação de Spearman. Os resultados demonstraram correlação positiva de ciclina D1 com escore de Gleason (p<0,05), com volume prostático (p=0,01) e uma tendência a correlação positiva com invasão perineural (p=0,07). Não houve correlação estatística entre ciclina D1 e o aumento de PSA, assim como outros achados histopatológicos. As biópsias prostáticas com agulha apresentaram subestimação em 40% dos casos para escore de Gleason e de 62,3% dos casos para lateralidade tumoral quando comparadas a prostatectomia radical. Já que as taxas de subestimação de escore de Gleason e lateralidade tumoral são relativamente altas e visto a urgência em se padronizar novos biomarcadores para o câncer prostático, sugerimos que ciclina D1 pode ser utilizada como biomarcador em patologia cirúrgica da próstata auxiliando numa gradação histológica mais precisa em biópsias com agulha colaborando para melhor vigilância e escolha terapêutica. / Prostate cancer is the most common malignant tumor in men older than 50 years, except for skin tumors. In Brazil it is estimated for the year 2012 about 60,180 new cases of this type of neoplasm. Tumor markers allow to cancer screening, differential diagnosis between a benign and malignant, assessment of prognosis and therapeutic monitoring, and detection of tumor recurrence. Among these tumor markers, has been given much attention for proteins that mediate and participate in cell cycle progression. Cyclin D1 is a short-lived nuclear protein that is destroyed by the ATP ubiquitin dependent pathway, and is involved in the transition of cell cycle G1 phase (resting) to the S phase (synthesis) cells both in normal and neoplastic cells. The overexpression of cyclin D1 removes the normal regulation of cell cycle causing uncontrolled cell proliferation, abnormal growth of tissues and transformation to a neoplastic phenotype, acting as an oncogene. In the present work we studied the expression of cyclin D1 in prostate adenocarcinomas, and to evaluate the relationship of this protein with epidemiologic factors, clinical and histopathological features. Additionally comparison was also made of Gleason score and laterality between tumor biopsies and prostate needle radical prostatectomies. In the assay for cyclin D1 were 85 cases analyzed by immunohistochemistry (IHC) of material from radical prostatectomies diagnosed with prostate adenocarcinoma between the years 2005 and 2010 at our institution. The evaluation method utilized were light microscopy and semi-quantitative score, comparing the cyclin D1 expression with clinical, epidemiological and histopathological features using Fisher\'s exact test, chi square test, Mann-Whitney test, ROC curve and Spearman correlation. The results showed a positive correlation of cyclin D1 with Gleason score (p <0.05), prostate volume (p = 0.01) and a trend toward positive correlation with perineural invasion (p = 0.07). There was no statistical correlation between cyclin D1 and increased PSA, as well as other histopathologic features. Prostate needle biopsies showed underestimation in 40% of cases for Gleason score and 62.3% of cases for tumor laterality when compared to radical prostatectomy. Since the rates of underestimation of Gleason score and tumor laterality are relatively high and the urgency to standardize new biomarkers for prostate cancer, we suggest that cyclin D1 may be used as biomarkers in surgical pathology of the prostate assisting more accurate histological grading in needle biopsies and collaborating for better surveillance and therapeutic choice.

Diversas são as formas de tratamento do câncer de próstata, com resultados oncológicos e complicações variadas . A crioablação foi proposta nos anos 60 e com a evolução dos métodos de imagem a técnica ressurgiu; 44 pacientes, divididos em 3 grupos (portadores de tumor de alto risco, tumores de baixo risco e falha de tratamento após radioterapia) foram submetidos a crioterapia por via percutânea transperineal. Sobrevida livre de doença foi de 87% no grupo baixo risco, 34% no grupo alto risco e 58% no grupo de resgate após falha de radioterapia. A complicação mais freqüente foi disfunção erétil (94,5%); obstrução infravesical ocorreu em 9 pacientes (20,4%); 6 pacientes (13,6%) apresentaram algum grau de incontinência urinária. Não houve nenhum caso de fístula uretroretal ou mortalidade relacionada ao procedimento / There are several treatments for prostate cancer with an assorted oncologic results and complications. Cryoablation was proposed in the 60 and the improvement of radiological techniques allowed the perineal percutaneous treatment; 44 patients divided into three groups (high risk tumors, low risk tumors and patients with recurrent prostate cancer following radiotherapy) were submitted to perineal percutaneous prostate cryoablation. Biochemical-free survival was 87% in low risk group, 34% in the high-risk group and 58% in salvage cryoablation. Erectile dysfunction was the most frequent complication (94,5%); Infravesical obstruction occurred in 20,4% of the patients and six (13,6%) developed urinary incontinence. There were no urethrorectal fistulae or mortality related to the procedure

Cheng, Cho Yiu. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2011. / Includes bibliographical references (leaves 186-217). / Abstracts in English and Chinese. / Acknowledgements --- p.1 / Abstract of thesis --- p.2 / Abstract of thesis in Chinese --- p.7 / Presentation attended --- p.9 / Chapter Chapter 1: --- Introduction and Background --- p.13 / Chapter 1.1 --- Anatomy and functions of human prostate gland --- p.13 / Chapter 1.2 --- Worldwide epidemiology of prostate cancer --- p.15 / Chapter 1.3 --- Prostate cancer stages and treatments in clinic --- p.21 / Chapter 1.4 --- Introduction to nuclear receptors --- p.23 / Chapter 1.5 --- Nuclear receptor structure --- p.24 / Chapter 1.6 --- Nuclear receptors nomenclature and classification --- p.28 / Chapter 1.7 --- Mode of action for nuclear receptors --- p.34 / Chapter 1.8 --- Co-regulators of nuclear receptors --- p.35 / Chapter 1.9 --- Nuclear receptors related to prostate cancer --- p.43 / Chapter Chapter 2: --- Aim of study and experimental design --- p.59 / Chapter 2.1 --- Aim of study --- p.59 / Chapter 2.2 --- In vitro cell lines models used in the study --- p.60 / Chapter Chapter 3: --- Materials and methods --- p.64 / Chapter 3.1 --- Apparatus and preparation throughout the study --- p.64 / Chapter 3.2 --- Cells culture --- p.64 / Chapter 3.3 --- RNA extraction --- p.67 / Chapter 3.4 --- Reverse transcription --- p.68 / Chapter 3.5 --- Primers specificity checking --- p.69 / Chapter 3.6 --- Real time quantitative polymerase chain reaction --- p.84 / Chapter 3.7 --- Data analysis --- p.90 / Chapter Chapter 4: --- Results --- p.92 / Chapter 4.1 --- Expression of nuclear receptors transcripts in each prostatic cell lines used --- p.92 / Chapter 4.2 --- Expression of nuclear receptor transcripts in immortalized prostatic epithelial BPH-1 and BPH-1 derived cell lines model --- p.116 / Chapter 4.3 --- Expression of nuclear receptor transcripts in androgen-dependent and androgen-independent classical prostatic cancer cell lines model --- p.121 / Chapter 4.4 --- Expression of nuclear receptor transcripts in androgen-independent and antiandrogen-resistant LNCaP derived cell lines model --- p.125 / Chapter Chapter 5: --- Discussion --- p.129 / Chapter 5.1 --- Special expression pattern of some nuclear receptors in the prostatic cell lines or prostatic cancer cell lines --- p.129 / Chapter 5.2 --- BPH-1 and BPH-1 derived cell lines model --- p.138 / Chapter 5.2.1 --- Prostatic cell lines model studying the transformation and invasion in prostate cancer (BPH-1 Snail & BPH-1 CAFTDs versus BPH-1) --- p.138 / Chapter 5.2.2 --- Prostatic cell lines model studying the transformation and invasion in prostate cancer (BPH-1 Snail & BPH-1 CAFTDs versus BPH-1 AR) --- p.159 / Chapter 5.3.3 --- classical prostatic cancer cell lines model --- p.162 / Chapter 5.3.1 --- Prostatic cancer cell lines model studying androgen-dependence and androgen-independence (DU145 & PC-3 versus LNCaP) --- p.163 / Chapter 5.4 --- LNCaP and LNCaP derived cell lines model --- p.170 / Chapter 5.4.1 --- Prostatic cancer cell lines model studying androgen-independence and antiandrogen-resistance (LNCaP-abl & LNCaP-BCs versus LNCaP) --- p.171 / Chapter Chapter 6: --- Conclusion --- p.179 / References --- p.186

研究背景和研究目的 / 前列腺癌是許多西方國家男性人群中最常見的惡性腫瘤。最新癌症統計結果表明，前列腺發病例和致死率在亞洲國家尤其是中國和香港地區呈迅猛上升趨勢(2009年，本港前列腺癌發病率列所有腫瘤發病率中第三位，致死率列第五位)。目前前列腺癌治療策略主要集中在拮抗雄激素信號通路。然而，臨床實踐表明，這種治療方式除了引起由於體內激素水平失調產生的一系列副作用之外，往往導致疾病進展到令人棘手的去勢治療無效階段。因此，從分子水平更為深入的理解前列腺癌疾病進展過程對於最終攻克前列腺癌具有重要的研究價值。雌激素相關受體是孤兒核受體的亞組之一，包括 α, β, γ三個亞型。該組受體在結構上與α亞型雌激素受體具有很高的同源性。已有研究表明，α亞型雌激素相關受體直接调控涉及氧化磷酸化，線粒體生物發生和脂肪酸氧化的相關基因表達，從而在細胞能量代謝調節中發揮至關重要作用。最新研究發現， α亞型雌激素相關受體的高表達在包括乳腺癌和前列腺癌在內的一系列腫瘤中與疾病的進展和不良預後高度相關。這提示該受體可能參與這些腫瘤的惡性進展。腫瘤細胞對低氧環境的耐受是實體腫瘤的標誌性表型之一，同時也有研究表明這一機制可能在癌細胞的惡性克隆選擇中發揮了重要作用。在眾多低氧耐受的機制中，細胞能量代謝方式轉換被研究人員看作重要的調節通路之一。考慮到前列腫瘤的低氧微環境以及α亞型雌激素相關受體在能量代谢過程的重要調節作用，有理由推測在該受體可能在前列腺癌細胞低氧耐受中發揮了積極的作用進而促進前列腺癌的惡性進展。 / 材料和方法 / 為了研究α亞型雌激素相關受體在前列腺癌細胞低氧耐受中的功能，本次研究採取了下列實驗方法：1）用免疫組化方法考察α亞型雌激素相關受體在人前列腺癌組織中的表達情況；2）用合適的前列癌細胞系建立α亞型雌激素相關受體穩定過表達細胞系同時研究這些穩轉細胞系的體外生長表型；）研究雌激素相關受體穩定過表達細胞系在低氧环境下的體外生長表型；）研究雌激素相關受體穩定過表達細胞系在免疫缺陷小鼠中的致瘤能力同時用免疫組化方法考察其腫瘤血管生成情況；）用定量 PCR和免疫印跡(Western blot)方法檢測低氧誘導因子-1α亞基(HIF-1α)及其信號通路中相關基因在α亞型雌激素相關受體穩定過表達細胞系中的表達水平，同時用雙螢光素酶報告基因方法考察α亞型雌激素相關受體對低氧誘導因子‐1(HIF-1)靶基因啟動子的轉錄激活效應；5）用 shRNA介導的基因阻斷的方法進一步考察α亞型雌激素相關受體對前列腺癌細胞低氧耐受的影響；6）通過觀考察用α亞型雌激素相關受體選擇性抑製劑 XCT790處理細胞對其在低氧環境下的體外生長情況的作用，進一步闡明 α亞型雌激素相關受體對前列腺癌細胞低氧耐受的影響；7）用免疫印跡 (Western blot)，免疫共沉澱 (Co-IP)和熒光能量共振轉移(FRET)分析的方法考察α亞型雌激素相關受體對低氧誘導因子‐1α亞基表蛋白表達和穩定性以及對低氧誘導因子 -1信號通路的影響。 / 結果 / 本研究所得得到的結果簡要總結如下：1）α亞型雌激素相關受體在前列癌組織中的免疫反應性呈現隨著惡性程度升高而增加的趨勢；2）α亞型雌激素相關受體在人前列腺癌細胞系 LNCaP中的過表達能提升其在常氧和低氧環境下的體外細胞增殖，細胞集落形成，細胞對胞外基質的粘附以及細胞侵襲能力； 3) α亞型雌激素相關受體在人前列腺癌細胞系 LNCaP中的過表達能促進其體內腫瘤形成及腫瘤血管生成； 4）過表達 α亞型雌激素相關受體能上調低氧誘導因子-1α亞基的蛋白水平並提高其轉錄活性；5）shRNA介導的α亞型雌激素相關受體 mRNA阻斷可以削弱人前列腺癌細胞系 LNCaP細胞在低氧環境下的體外生長能力；6）在体外用α亞型雌激素相關受體選擇性抑製劑 XCT790处理人前列腺癌細胞系 LNCaP細胞可能通過減少低氧誘導因子‐1α亞基蛋白表達水平從而抑制其在低氧環境下的細胞生長能力；7）α亞型雌激素相關受體可以直接與低氧誘導因子-1α亞基相互作用，並且這種相互作用可能有助於抑制低氧誘導因子-1 α亞基的蛋白降解。 / 結論 / 本研究獲得結果提示，α亞型雌激素相關受體可能通過提高低氧誘導因子-1α亞基的蛋白水平及激活低氧誘導因子-1信號通路從而促進前列腺癌細胞在低鹽環境下的細胞生長能力。体外用 shRNA介導的α亞型雌激素相關受體 mRNA阻斷方法和α亞型雌激素相關受體選擇性抑製劑处理都有可能通過阻止低氧誘導因子‐1α亞基以削弱前列腺癌細胞在低鹽環境下的細胞生長能力。同時， α亞型雌激素相關受體能直接與低氧誘導因子-1 α亞基相互作用而這種相互作用有可能有助於抑制其蛋白降解，這些結果提示 α亞型雌激素相關受體可能在前列腺癌進展過程中的低氧耐受中發揮積極作用。 / Background and aims of study / Prostate cancer is the most common cancer in many Western counties among the male populations. Latest cancer statistics also show that its incidence and mortality rates are rapidly increasing in China and Hong Kong (Prostate cancer ranked the 3rd common cancer and 5th cancer causing death in Hong Kong in 2009). Current therapeutic strategies of prostate cancer mainly target to the antagonizing androgen signaling pathway, which usually drives the disease to the impasse of castration resistance albeit the side effects caused by the imbalance of hormone. The substantial clinical significance of prostate cancer is urgent to better understand the progression of this disease. Estrogen-related receptors (α,β,γ) are a subgroup of ligand-independent orphan nuclear receptors, which is constitutively activated without binding any physiological ligands and all share high homology with the estrogen receptor alpha (ER α) structurally. Previous studies indicates that ERR α plays a pivotal role in cellular energy home stasis regulation, target genes of which are involved in the procedures of oxidative phosphorylation, mitochondrial biogenesis and fatty acid oxidation. Recent studies reveals that high expression of ERR α may be useful as a poor prognostic marker in both hormone-dependent and hormone-independent cancers (including breast cancer and prostate cancer), which implicates this nuclear receptor may be involved in the advanced malignant progression of these cancers. Adaptation to hypoxia is one of the hallmark features of solid tumors and it is conceived to play an important role in malignant clonal selection of cancer cells. Among the diverse mechanisms on cellular hypoxia adaptation, energy metabolism reprogramming is characterized and considered as a critical regulatory pathway. Given the hypoxic microenvironment of prostate cancer and the energy regulatory role of ERR α, it is hypothesized that ERR α might play an active role in the cellular hypoxic adaptation of prostate cancer hence advancing the progre sion of this disease. / Materials and methods / To investigate the functional significance of ERR α in cellular hypoxic adaptation of prostate cancer, the following experimental approaches were employed and performed in my thesis study: 1) to survey the expression pattern of ERR α in human prostate cancer tissues by immunohistochemical staining; 2) to generate ERR α-stable expressing cell lines in selected prostate cancer cell lines and functionally characterize their in vitro phenotypes under normoxia condition; 3) to characterize in vitro hypoxic-response phenotypes of ERR α-infectants; 4) to determine the tumorigenicity of ERR α-infectants in immuno-deficient SCID mice and to investigate their tumor angiogenesis by immunohistochemical staining; 5) to determine the HIF-1α signal cohort in ERR α-infectants by both RT-PCR and immuno blot analysis and to investigate the transactivation effect of ERR α on HIF-1 targeting genes promoters by dual luciferase reporter assay; 6) to further characterize the hypoxic adaptation phenotypes induced by ERR α transduction using shRNA-mediated gene knockdown approach; 7) to further elucidate the effect of ERR α on the hypoxic cell growth regulation of prostate cancer by treating ERR α-infectants with an ERR α-selective antagonist XCT790; 8) to further investigate the mechanisms via which ERR α interferes with the protein expression or stabilization of HIF-1α as well as HIF-1 signal cohort using immuno blot analysis, immunoprecipitation assays and fluorescence resonance energy transfer (FRET) analysis. / Results / My results are briefly summarized as follows: 1) ERR α exhibited an increased immuno expression pattern in high-grade prostate cancer; 2) Ectopic expression of ERR α in LNCaP prostate cancer cell line could promote its in vitro cell proliferation, clonal formation, cell-extracellular matrix attachment and cell invasion capacities under both normoxic and hypoxic conditions; 3) Ectopic expression of ERR α in LNCaP prostate cancer cell line could promote its in vivo tumorigenicity and tumor angiogenesis; 4) Overexpression of ERR α could up-regulate protein level of hypoxia regulatory transcriptional factor-1(HIF-1) α subunit (HIF1-α) and enhance its transcriptional activity; 5) mRNA knock-down of ERR α could attenuate in vitro cell growth capacity of LNCaP prostate cancer cell line under hypoxic condition; 6) Treatment with an ERR α specific antagonist XCT790 could inhibit in vitro hypoxic cell growth of LNCaP cells via its effect on decreasing the protein level of HIF-1α; 7) ERR α could physically interact with HIF-1α and such ERR α-HIF1-α interaction might help to inhibit protein degradation of HIF-1α. / Conclusion / The results obtained in this study indicated that ERR α could promote the hypoxic cell growth of prostate cancer via its enhancing the protein level of HIF-1α and activation of HIF-1 signal cohort. Both treatment with ERR α selective antagonist and down-regulating of ERR α by shRNA-mediated gene knockdown approach could attenuate the hypoxia adaptation of prostate cancer cells, which might be mediated by their suppression of the protein level of HIF1α. ERR α could directly interact with HIF-1α and such interaction might help to suppress the protein degradation of HIF1α, suggesting that ERR α may play an active role in hypoxic adaptation in advancing of prostate cancer. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Zou, Chang. / Thesis (Ph.D.)--Chinese University of Hong Kong, 2012. / Includes bibliographical references (leaves 138-160). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Abstract also in Chinese. / ABSTRACT --- p.i / ACKNOWLEDGEMENTS --- p.viii / PUBLICATIONS --- p.ix / CONTENTS --- p.x / ABBREVIATIONS --- p.xiii / Chapter CHAPTER 1 --- Introduction --- p.1 / Chapter 1.1 --- Prostate cancer --- p.2 / Chapter 1.1.1 --- Epidemiology --- p.2 / Chapter 1.1.2 --- Risk factors --- p.3 / Chapter 1.1.3 --- Patho-physiology --- p.6 / Chapter 1.1.4 --- Diagnosis and treatment --- p.8 / Chapter 1.2 --- Androgen,androgen receptor and prostate cancer --- p.10 / Chapter 1.2.1 --- Androgen and androgen receptor --- p.10 / Chapter 1.2.2 --- Castration Resistance Prostate Cancer (CRPC) --- p.12 / Chapter 1.2.2.1 --- Overexpression of AR --- p.13 / Chapter 1.2.2.2 --- Increasing sensitivity to and rogen --- p.13 / Chapter 1.2.2.3 --- AR mutation --- p.14 / Chapter 1.2.2.4 --- Deregulation of AR regulator factors --- p.15 / Chapter 1.2.2.5 --- Outlaw pathway --- p.15 / Chapter 1.2.2.6 --- AR-independent pathway --- p.16 / Chapter 1.3 --- Estrogen and prostate cancer --- p.17 / Chapter 1.3.1 --- Overview of estrogen and estrogen receptors --- p.17 / Chapter 1.3.2 --- Estrogen signaling pathway andprostatecancer --- p.18 / Chapter 1.4 --- Nuclear receptors --- p.20 / Chapter 1.4.1 --- Overview of NRs superfamily --- p.20 / Chapter 1.4.2 --- Classification --- p.21 / Chapter 1.4.3 --- NRs as therapeutic targets for cancer treatment --- p.23 / Chapter 1.5 --- Estrogen-related receptors --- p.25 / Chapter 1.5.1 --- NR3B subgroup --- p.25 / Chapter 1.5.2 --- Isoforms --- p.26 / Chapter 1.5.3 --- Structure --- p.27 / Chapter 1.5.4 --- Ligand --- p.28 / Chapter 1.5.5 --- Co-regulators --- p.31 / Chapter 1.5.6 --- Tissue-specific expression pattern and identifiedfunction --- p.32 / Chapter 1.5.6.1 --- Tissue-specific expression pattern --- p.32 / Chapter 1.5.6.2 --- Identified physiological function of ERRs --- p.33 / Chapter 1.5.7 --- ERRs and cancer --- p.35 / Chapter 1.5.7.1 --- ERRβ/γ and cancer --- p.35 / Chapter 1.5.7.2 --- Expression of ERRα in cancer --- p.37 / Chapter 1.5.7.3 --- Identified functional roles of ERRα in cancer --- p.40 / Chapter 1.5.7.4 --- Regulation of ERRα in cancer cells --- p.42 / Chapter 1.6 --- Hypoxiaadaptation andcancer --- p.47 / Chapter 1.6.1 --- HIFs isoforms and structure --- p.47 / Chapter 1.6.2 --- Structure --- p.48 / Chapter 1.6.3 --- Regulation of HIF-1α expression --- p.49 / Chapter 1.6.3.1 --- Regulation of HIF-1α mRNA transcription --- p.49 / Chapter 1.6.2.2 --- Regulation of HIF-1α mRNA transcription --- p.50 / Chapter 1.6.2.3 --- O₂-dependent regulation of stability of HIF-1α protein --- p.51 / Chapter 1.6.2.4 --- O₂-independent regulation of HIF-1α --- p.52 / Chapter 1.6.2.5 --- Genetranscriptional regulation role of HIFs --- p.54 / Chapter 1.6.3 --- HIFs and cancer --- p.55 / Chapter 1.6.3.1 --- Overview --- p.55 / Chapter 1.6.3.2 --- Expression of HIF-1α in cancer progression --- p.55 / Chapter 1.6.3.2 --- Functional roles of HIF-1α in cancer progression --- p.56 / Chapter CHAPTER 2 --- Aims of study --- p.58 / Chapter CHAPTER 3 --- Materials and methods --- p.61 / Chapter 3.1 --- Cell lines and cell culture --- p.62 / Chapter 3.2 --- Human Prostatic Tissues --- p.64 / Chapter 3.3 --- RNA isolation and Reverse transcriptional-PCR --- p.64 / Chapter 3.3.1 --- Total RNA extraction --- p.64 / Chapter 3.3.2 --- Reverse transcription reaction --- p.65 / Chapter 3.3.3 --- Polymerase Chain Reaction for gene expression detection --- p.66 / Chapter 3.4 --- Plasmids construction --- p.69 / Chapter 3.4.1 --- Genomic DNA extraction --- p.69 / Chapter 3.4.2 --- PCR for cloning and sub-cloning --- p.70 / Chapter 3.4.3 --- PCR for mutant generation --- p.70 / Chapter 3.4.4 --- Restriction enzymes cut and ligation --- p.71 / Chapter 3.5 --- Antibody and reagents --- p.73 / Chapter 3.6 --- Immunohistochemistry --- p.74 / Chapter 3.7 --- Western Blot Analysis --- p.75 / Chapter 3.7.1 --- Protein extraction --- p.75 / Chapter 3.7.2 --- Electrophoresis, Protein blotting and Colorimetric detection --- p.76 / Chapter 3.8 --- Retroviral transduction and generation of ERRα poolandstable clones --- p.77 / Chapter 3.9 --- In vitro Cell Growth Assays --- p.77 / Chapter 3.9.1 --- Cell counting --- p.77 / Chapter 3.9.2 --- 5-Bromodeoxyuridine (BrdU) incorporation assay --- p.78 / Chapter 3.9.3 --- MTT assay --- p.79 / Chapter 3.9.4 --- In vitro clonal formation assay --- p.79 / Chapter 3.10 --- Cell attachment assay --- p.80 / Chapter 3.11 --- Transwell cell invasion assay --- p.81 / Chapter 3.12 --- In vivo tumorigenicity assay --- p.81 / Chapter 3.13 --- RNA interference --- p.82 / Chapter 3.14 --- Transient Transfection and Luciferase Reporter Assay --- p.83 / Chapter 3.15 --- Immuno-precipitation (IP) assay --- p.84 / Chapter 3.16 --- Fluorescence Resonance Energy Transfer (FRET) detection --- p.85 / Chapter 3.17 --- In vitro treatment with XCT790, cycloheximide and MG-132 --- p.86 / Chapter CHAPTER 4 --- Reuslts --- p.88 / Chapter 4.1 --- ERRα exhibits an increased expression pattern in high grade prostate cancer --- p.89 / Chapter 4.2 --- Ectopic expression of ERRα in LNCaP prostate cancer cell line can promote its in vitro cell proliferation, clonal formation, cell attachment and cell invasion capacity under normoxic condition --- p.91 / Chapter 4.3 --- Ectopic expression of ERR α in LNCaP prostate cancer cell line can promote its in vitro cell proliferation, clonal formation, cell attachment and cell invasion capacities under hypoxic condition --- p.94 / Chapter 4.4 --- Ectopic expression of ERR α in LNCaP prostate cancer cells can promote their in vivo tumorigenicity and tumor angiogenesis. --- p.97 / Chapter 4.5 --- Overexpression of ERRα can up‐regulate protein level of HIF-1α and enhance its transcriptional activity --- p.99 / Chapter 4.6 --- mRNA Knock-down of ERRα can attenuate in vitro cell growth of LNCaP prostate cancer celll line under hypoxic condition --- p.107 / Chapter 4.7 --- Treatment with an ERRα specific antagonist XCT790 can inhibit in vitro hypoxic cell growth of LNCaP cells via its effect on decreasing the protein level of HIF-1α --- p.110 / Chapter 4.8 --- ERRα can physically interact with HIF-1α and such ERRα-HIF-1α interaction helps to inhibit protein degradation of HIF-1α --- p.114 / Chapter CHAPTER 5 --- Discussion --- p.119 / Chapter CHAPTER 6 --- Summary --- p.134 / References --- p.138

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.