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
| Identifer | oai:union.ndltd.org:cuhk.edu.hk/oai:cuhk-dr:cuhk_328238 |
| Date | January 2012 |
| Contributors | Zou, Chang., Chinese University of Hong Kong Graduate School. Division of Biomedical Sciences. |
| Source Sets | The Chinese University of Hong Kong |
| Language | English, Chinese |
| Detected Language | English |
| Type | Text, bibliography |
| Format | electronic resource, electronic resource, remote, xv, 160 leaves : ill. (some col.) |
| Rights | Use of this resource is governed by the terms and conditions of the Creative Commons “Attribution-NonCommercial-NoDerivatives 4.0 International” License (http://creativecommons.org/licenses/by-nc-nd/4.0/) |
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