研究背景與研究目的 / 細胞衰老是指細胞進入不可逆的永久化的生長停滯狀態。目前，細胞衰老作為重要的抑癌機制受到廣泛認可，对其相關信號通路的研究為腫瘤的靶向治療提供了新的依據和策略。TLX核受體基因属于核受體亞家族2組E成員1，是一種孤兒受體。雞和老鼠TLX基因最初作為果蠅末端/间隙基(tailless) 的同源基因而被發現，而人TLX 基因是在檢索惡性淋巴癌中的抑癌細胞而從人胚胎的腦cDNA文庫中克隆出來的。TLX基因敲除的轉基因老鼠的研究表明TLX基因對維持胚胎腦和成体腦神經幹細胞的分裂增殖起重要作用。最近的研究發現，TLX在臨床神經胶质瘤組織中高表達。並且，在轉基因鼠中，TLX的高表達会引起神經幹細胞的大量增殖而形成腦腫瘤，提示TLX可能參與腦腫瘤的發生和發展。但是，TLX对包括前列腺癌在内的人類惡性腫瘤的發生發展中所起的功能及作用機制尚不清楚。表達譜研究發現，TLX在前列腺細胞中的表達水平高於永生化的正常前列腺上皮細胞的表達，並且，TLX在臨床惡性程度高的前列腺癌中呈高表達趨勢，預示TLX可能參與促進前列腺癌的惡性進展。因此本研究的主要目的是TLX在前列腺癌細胞中的功能研究。 / 研究材料與方法 / 為了研究TLX對前列腺癌細胞生長的影響以及相關機制，本論文主要採用以下方法：1）運用免疫組化的方法檢測TLX在臨床前列腺癌組織中的表達，並應用實時螢光定量PCR方法檢測TLX在永生化的非惡性前列腺上皮細胞以及前列腺癌細胞株中的表達；2）根據不同的p53表達狀態選擇雄激素依賴（LNCaP）和雄激素非依賴（PC-3, DU145）的前列腺癌細胞株，分別采用慢病毒感染和逆轉錄病毒感染的方法建立TLX-敲除和TLX-過表達的細胞株，並研究這些穩轉細胞系離體和在體的生長表型（包括檢測細胞生長，細胞週期，細胞衰老，細胞的遷移和侵染，化療藥物抗性，缺氧耐受性以及體內成瘤能力）；3）採用檢測β-半乳糖苷酶活性的方法檢測TLX穩轉系細胞在衰老因素誘導和非誘導狀態下TLX缺失和高表達對細胞衰老的影響；4）采用免疫印跡（western blot）的方法檢測TLX穩轉系細胞中參與細胞衰老的關鍵蛋白的表達情況；5）利用雙螢光素酶報告基因方法和染色質免疫沉澱技術，研究TLX對靶基因的調控；6）構建TLX缺失變異體（△ZF1 和 △LBD-AF2），在前列腺細胞系和非前列腺細胞系中外源性表達相應的變異體進一步驗證TLX的功能。 / 結果 / 本論文研究結果總結如下：1）TLX在前列腺癌細株中和惡性程度高的前列腺癌組織中高表達；2）在前列腺癌中進行TLX基因敲除能顯著抑制細胞體外和體內的生長並誘導前列腺癌細胞的衰老；3）相反，TLX的過表達能促進前列腺癌細胞體外和體內的惡性生長，包括促進細胞的錨定和非錨定性生長、促進細胞的遷移與侵染、增強細胞缺氧耐受、對化療藥物抗性、以及增強細胞異位移植瘤的成瘤能力；4）TLX 的高表達抑制了前列腺癌細胞衰老，並保護細胞免受多柔比星誘導的細胞衰老以及持續性激活的癌基因H-RAS（H-RAS{U+1D33}¹²{U+2C7D}）誘導的細胞衰老；5）TLX可以結合到p21{U+1D42}{U+1D2C}{U+A7F1}¹/{U+A7F0}{U+1D35}{U+1D3E}¹基因（其後縮寫為p21）的啟動子序列並抑制p21的啟動子的轉錄活性，並且在TLX-過表達細胞中外源性高表達p21能誘導前列腺癌細胞重新進入衰老狀態；6）TLX也能結合到SIRT1基因的啟動子序列並激活SIRT1的轉錄活性，在TLX-過表達細胞中對SIRT1進行基因沉默能誘導這些細胞的再次衰老；7）TLX介導的衰老抑制效應以及對其靶基因的轉錄調控作用需要完整的DNA-結合域以及配體結合域，對TLX兩個區域的缺失變異影響TLX在前列腺細胞和非前列腺細胞中的生理功能及轉錄調控活性。 / 結論 / 本論文的研究結果提示TLX通過抑制前列腺癌細胞的衰老在前列腺癌發生發展過程中起重要作用，並且這種衰老抑制作用是通過介導p21基因的轉錄抑制以及對SIRT1基因的轉錄激活而實現的。此研究首次證實了TLX在前列腺癌中高表達，並且TLX能夠抑制前列腺癌細胞的衰老從而促進前列腺癌的發生發展，提示TLX有可能成為前列腺癌治療潛在的重要靶點。 / Background and aims of the study / Cellular senescence represents an irreversible form of permanent cell-cycle arrest and it acts a key process of tumor suppression, while targeting to pathways involved in this process can provide potential and promising therapeutic strategies to cancer treatments. TLX belongs to the NR2E1 orphan nuclear receptor subfamily. The chicken and mouse TLX genes were initially isolated as a vertebrate homolog to the Drosophila terminal-gap gene tailless (tll), while the human TLX was cloned from a fetal brain cDNA library in a search for putative tumor suppressor genes in lymphoid malignancies. Functional studies in transgenic mouse model of TLX-knockdown show that TLX plays important regulatory roles in the maintenance and self-renewal control of both embryonic and adult neural stem cells. Recent studies of transgenic mice with TLX overexpression combined with its expression studies in human clinical gliomas revealed that TLX is overexpressed in primary human glioblastomas and its dysregulation may contribute to the initiation and development of some brain tumors. However, the exact functional contributions of TLX and the involved mechanism(s) in human malignancies, including prostate cancer, are still far from clear. In an expression profile study, it was demonstrated that TLX exhibited an up-regulated expression pattern in many prostate cancer cell lines and also the high-grade clinical prostate cancer, suggesting that TLX might play a positive regulatory role in the advanced progression of prostate cancer. The overall aim of this study was to elucidate the functional role of TLX in prostate cancer cell growth. / Materials and methods / In order to elucidate the functional roles of TLX in prostate cancer growth and the involved mechanisms, the following experiments were conducted: 1) To investigate and determine the expression pattern of TLX in clinical prostatic tissues by immunohistochemistry, and to survey the expression profile of TLX in a panel of prostatic immortalized epithelial and prostate cancer cell lines by quantitative real-time PCR analysis; 2) To generate stable TLX-knockdown prostate cancer cells by lentiviral transduction and TLX-stable expressing cells by retroviral transduction in both hormone-sensitive (LNCaP) and -insensitive (DU145 and PC-3) prostate cancer lines with different expression status of p53; and to conduct growth phenotype characterization studies (including cell growth, cell cycle, cellular senescence, cell migration and invasion, resistance to chemotherapy drugs, hypoxic cell growth assays, and tumorigenesis) on these TLX-transfectants in vitro and in vivo; 3) To characterize cellular senescence phenotype of TLX-infectants by senescence-associated β-galactosidase (SA-β-Gal) staining method with or without senescence inducers; 4) To investigate the expression status of markers involved in cellular senescence in TLX-infectants by immunoblotting; 5) To demonstrate the transcriptional regulation targets of TLX by dual-luciferase reporter assay and chromatin immunoprecipitation (ChIP) assay; 6) To confirm the cellular function of TLX in prostatic and non-prostatic cells expressing different TLX deletion mutants (△ZF1 and △LBD-AF2). / Results / Results obtained in this study are summarized as follows: 1) TLX displayed an increased expression pattern in many prostate cancer cell lines and also high-grade (Gleason score ≥ 7) prostate cancer tissues; 2) Depletion of TLX mRNA by RNA interference dramatically suppressed in vitro and in vivo tumor cell growth and triggered cellular senescence (SA-β-Gal histochemical marker) in prostate cancer cells; 3) On the contrary, TLX overexpression significantly enhanced multiple advanced malignant growth capacities (including enhanced anchorage-dependent and -independent cell growth, cell migration and invasion, hypoxia adaptation, resistance to chemotherapy drug Doxorubicin as well as in vivo tumorigenicity) in prostate cancer cells; 4) TLX overexpression significantly suppressed cellular senescence and protected cells against doxorubicin-induced or oncogenic H-RAS (H-RAS{U+1D33}¹²{U+2C7D})- induced senescence; 5) TLX could directly bind to p21{U+1D42}{U+1D2C}{U+A7F1}¹/{U+A7F0}{U+1D35}{U+1D3E}¹ gene (hereafter p21) promoter and repress the transcriptional activity of p21 promoter, while ectopic restoration of p21 expression in TLX-overexpressed cells could rescue cellular senescence with enhanced SA-β-Gal staining; 6) protein deacetylase SIRT1 gene was also activated by TLX through its direct transcriptional regulation, while knockdown of SIRT1 in TLX-overexpressed cells could rescue cellular senescence; 7) TLX-induced suppression of cellular senescence and also its direct gene regulation would require an intact DBD and LBD domain, as truncated deletion of DBD or LBD domain could both abolish the cellular function and transcriptional activity of TLX in prostatic and non-prostatic cells. / Conclusions / The results obtained in this study suggested that TLX could play a positive growth regulatory or tumor-promoting role in prostate cancer development by its suppression of cellular senescence and this senescence suppression was mediated via its direct transcriptional regulation of both p21 (repression) and SIRT1 (transactivation) genes. Moreover, this study also showed for the first time that TLX, which was overexpressed in prostate cancer tissues, might function to suppress premature senescence in prostate cancer progression and also targeting to TLX could be a potential therapeutic approach for prostate cancer treatment. / 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. / Wu, Dinglan. / Thesis (Ph.D.)--Chinese University of Hong Kong, 2012. / Includes bibliographical references (leaves 135-151). / Abstract also in Chinese. / Thesis /Assessment Committee --- p.I / ABSTRACT --- p.II / 摘 要 --- p.VI / ACKNOWLEDGEMENT --- p.IX / PUBLICATIONS RELATED TO THIS THESIS --- p.XI / CONTENTS --- p.XII / ABBREVIATION --- p.XV / Chapter CHAPTER 1 --- INTRODUCTION --- p.1 / Chapter 1.1 --- Prostate cancer --- p.2 / Chapter 1.1.1 --- Epidemiology --- p.2 / Chapter 1.1.2 --- Nature history --- p.4 / Chapter 1.1.3 --- Androgen Axis prostate cancer --- p.7 / Chapter 1.1.3.1 --- Androgen receptor --- p.7 / Chapter 1.1.3.2 --- Function of the androgen receptor in prostate cancer --- p.7 / Chapter 1.1.3.3 --- Mechanisms of CRPC progression --- p.8 / Chapter 1.1.3.4 --- Androgen receptor pathway-directed therapies --- p.10 / Chapter 1.1.4 --- Treatment of prostate cancer --- p.11 / Chapter 1.2 --- Cellular senescence --- p.13 / Chapter 1.2.1 --- What is senescence --- p.13 / Chapter 1.2.1.1 --- Replicative cellular senescence --- p.13 / Chapter 1.2.1.2 --- Oncogene induced senescence (OIS) --- p.15 / Chapter 1.2.1.3 --- Tumor suppressor loss-induced senescence --- p.17 / Chapter 1.2.2 --- Establishment of cellular senescence --- p.19 / Chapter 1.2.3 --- The p16/pRb and ARF/p53/p21 pathway of senescence induction --- p.21 / Chapter 1.2.3.1 --- p16/pRb senescence pathway --- p.22 / Chapter 1.2.3.2 --- ARF/p53/p21 senescence pathway --- p.23 / Chapter 1.2.4 --- Markers of senescence --- p.24 / Chapter 1.2.4.1 --- Cell cycle arrest and morphology --- p.24 / Chapter 1.2.4.2 --- Senescence-associated β-galactosidase --- p.25 / Chapter 1.2.4.3 --- p16/pRb and p53/p21 pathways --- p.26 / Chapter 1.2.4.4 --- γ-H2AX staining as a marker for DNA damage --- p.27 / Chapter 1.2.4.5 --- Senescence-associated heterochromatin foci (SAHF) --- p.27 / Chapter 1.2.5 --- Pro-senescence therapy for cancer treatment --- p.29 / Chapter 1.2.5.1 --- Why pro-senescence therapy --- p.29 / Chapter 1.2.5.2 --- Critical factors of pro-senescence therapy --- p.31 / Chapter 1.2.5.3 --- Strategies of senescence induction --- p.32 / Chapter 1.2.5.4 --- Targeting to senescence-associated secretory phenotype (SASP) --- p.38 / Chapter 1.2.6 --- Future direction --- p.40 / Chapter 1.3 --- TLX --- p.41 / Chapter 1.3.1 --- Nuclear receptor --- p.41 / Chapter 1.3.2 --- Identification of tailless/TLX --- p.42 / Chapter 1.3.3 --- Tailless in drosophila --- p.43 / Chapter 1.3.4 --- Functional role of tll/TLX --- p.45 / Chapter 1.3.4.1 --- Role of tll/TLX in brain development --- p.45 / Chapter 1.3.4.2 --- Role of tll/TLX in visual system developments --- p.46 / Chapter 1.3.4.3 --- Role of TLX in neural stem cell self-renewal --- p.47 / Chapter 1.3.5 --- Target genes of TLX --- p.49 / Chapter 1.3.6 --- Transcriptional regulation of tll/TLX --- p.51 / Chapter 1.3.7 --- TLX in cancer --- p.52 / Chapter CHAPTER 2 --- STUDY AIMS --- p.54 / Chapter CHAPTER 3 --- MATERIALS AND METHODS --- p.57 / Chapter 3.1 --- Human prostatic tissues and Immunohistochemistry --- p.58 / Chapter 3.2 --- Cell lines and cell cultures --- p.59 / Chapter 3.3 --- Antibody and reagents --- p.63 / Chapter 3.3.1 --- Generation of rabbit anti-TLX polyclonal antibody --- p.63 / Chapter 3.3.2 --- Commercial antibody --- p.64 / Chapter 3.4 --- RNA isolation and Reverse transcriptional-PCR --- p.65 / Chapter 3.4.1 --- RNA isolation --- p.65 / Chapter 3.4.2 --- Reverse transcription reaction (RT) --- p.66 / Chapter 3.4.3 --- Polymerase Chain Reaction (PCR) --- p.66 / Chapter 3.5 --- Western blotting --- p.68 / Chapter 3.5.1 --- Protein extraction --- p.68 / Chapter 3.5.2 --- Electrophoresis, Protein blotting and Colorimetric detection --- p.69 / Chapter 3.6 --- Plasmids construction --- p.70 / Chapter 3.6.1 --- PCR for sub-cloning --- p.70 / Chapter 3.6.2 --- PCR for mutant generation --- p.71 / Chapter 3.6.3 --- Restriction enzymes digestion and ligation --- p.72 / Chapter 3.7 --- Retroviral, lentiviral transduction and generation of TLX-stable cells --- p.73 / Chapter 3.8 --- RNA interference --- p.75 / Chapter 3.9 --- In vitro cell growth assay --- p.76 / Chapter 3.9.1 --- Cell counting --- p.76 / Chapter 3.9.2 --- MTT assay --- p.76 / Chapter 3.9.3 --- Soft agar assay for anchorage independent growth --- p.77 / Chapter 3.10 --- Cell cycle assay --- p.77 / Chapter 3.11 --- Cell invasion assay --- p.78 / Chapter 3.12 --- In vivo tumor growth assay --- p.78 / Chapter 3.13 --- In vitro and in vivo SA-β-Gal staining --- p.79 / Chapter 3.14 --- In vitro treatment with doxorubicin --- p.80 / Chapter 3.15 --- Transient Transfection and Luciferase Reporter Assay --- p.81 / Chapter 3.16 --- Chromatin immunoprecipitation (ChIP) assay --- p.82 / Chapter 3.16.1 --- Cross-linking and harvesting cells --- p.82 / Chapter 3.16.2 --- Cell lysis --- p.83 / Chapter 3.16.3 --- Sonication --- p.83 / Chapter 3.16.4 --- Immunoprecipitation --- p.83 / Chapter 3.16.5 --- Washing --- p.84 / Chapter 3.16.6 --- Elution --- p.85 / Chapter 3.16.7 --- Reverse cross-linking and DNA purification --- p.85 / Chapter 3.16.8 --- PCR --- p.86 / Chapter 3.17 --- Statistical analysis --- p.86 / Chapter CHAPTER 4 --- RESULTS --- p.87 / Chapter 4.1 --- TLX is up-regulated in prostate carcinoma and prostate cancer cell lines --- p.88 / Chapter 4.2 --- Knockdown of TLX suppresses in vitro cell growth and triggers cellular senescence in prostate cancer cells --- p.93 / Chapter 4.3 --- Knockdown of TLX inhibits in vivo tumor growth and induces cellular senescence of prostate cancer cells --- p.97 / Chapter 4.4 --- Ectopic expression of TLX enhances in vitro cell growth and multiple advanced malignant phenotypes in prostate cancer cells --- p.100 / Chapter 4.5 --- Ectopic expression of TLX suppresses cellular senescence in prostate cancer cells --- p.105 / Chapter 4.6 --- TLX suppresses cellular senescence via its direct transcriptional repression of p21{U+1D42}{U+1D2C}{U+A7F1}¹/{U+A7F0}{U+1D35}{U+1D3E}¹ gene --- p.110 / Chapter 4.7 --- TLX also suppresses cellular senescence via its transcriptional regulation of SIRT1 gene --- p.116 / Chapter CHAPTER 5 --- DISCUSSION --- p.121 / Chapter CHAPTER 6 --- SUMMARY --- p.131 / REFERENCES --- p.135

Xiao, Lijia. / Thesis (Ph.D.)--Chinese University of Hong Kong, 2013. / Includes bibliographical references (leaves 139-158). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Abstract also in Chinese.

Made available in DSpace on 2014-12-17T15:32:24Z (GMT). No. of bitstreams: 1 ThamaraMMB_DISSERT.pdf: 1856423 bytes, checksum: d77238963a3bf6663f56d36a18db57b6 (MD5) Previous issue date: 2014-02-27 / Coordena??o de Aperfei?oamento de Pessoal de N?vel Superior / Th17 cells have been strongly associated to the pathogenesis of inflammatory and autoimmune diseases, although their influence on the carcinogenesis is still little known, there are reports of anti-tumor and protumoral actions. The objective of this study is to research the presence of Th17 lineage in lip and tongue SCC, using the analysis of the immunoexpression of IL-17 and ROR?t, relating this immunoexpression with clinical and morphological findings in the attempt to better comprehend the role of these cells on the tumoral immunity of OSCCs. The results were submitted to non-parametric statistical tests with significance level of 5%. On the histomorphological analysis, it was observed the predominance of low level lesions on lip and high level lesions on tongue (p=0,024). It was not observed statistical significance between clinical stage and histological gradation of malignancy (p=0,644). For the immunohistochemical study, 5 random fields with greater immunoreactivity of the peritumoral inflammatory infiltrate were photomicrographed on the 400x magnification. It was done the count of lymphocytes which showed cytoplasmic and pericytoplasmic staining for the IL-17 cytokine as well as nuclear and cytoplasmic staining for ROR?t. It was observed statistical significance difference on the quantity of immunopositive lymphocytes to IL-17 between the groups of SCC of lip and tongue (p=0,028). For the ROR?t it was not observed statistical significance difference between the groups of SCC of lip and tongue (p=0,915). It was not observed statistical difference between the immunostaining of IL-17 and ROR?t with histological gradation of malignancy and clinical staging. The findings of this research suggest a possible anti-tumor role of IL-17 for cases of lip. The results of the analysis of the ROR?t are possibly due to the wide duality of the anti-tumor and protumoral role of the Th17 cells and their plasticity which, in the presence of different cytokines expressed on the tumor microenvironment, can alter its phenotype. / As c?lulas Th17 t?m sido fortemente associadas ? patogenia de doen?as autoimunes e inflamat?rias, por?m sua influ?ncia na carcinog?nese ainda ? pouco conhecida, havendo relatos de suas a??es tanto antitumorais quanto pr?-tumorais. O objetivo do presente trabalho foi pesquisar a presen?a da linhagem Th17 intratumoral em CCE de l?bio e l?ngua, atrav?s da an?lise da imunoexpress?o da IL-17 e do ROR?t, relacionando estes achados com dados cl?nicos e morfol?gicos na tentativa de melhor compreender o papel dessas c?lulas na imunidade tumoral dos CCEOs. Na an?lise histomorfol?gica, observou-se predom?nio de les?es de baixo grau em l?bio e de alto grau em l?ngua (p = 0,024). N?o foi observada signific?ncia estat?stica entre estadiamento cl?nico e grada??o histol?gica de malignidade (p = 0,644). Para o estudo imunoistoqu?mico, 5 campos aleat?rios com maior imunorreatividade do infiltrado inflamat?rio peritumoral foram fotomicrografados no aumento de 400x. Realizou-se a contagem de linf?citos que exibiram marca??o citoplasm?tica e pericitoplasm?tica para a citocina IL-17 bem como nuclear e citoplasm?tica para o ROR?t. Foi observada diferen?a estatisticamente significativa na quantidade de linf?citos imunopositivos para IL-17 entre os grupos de CCE de l?bio e l?ngua (p = 0,028). Para o ROR?t n?o foi observada diferen?a estatisticamente significativa entre os grupos de CCE de l?bio e l?ngua (p = 0,915). N?o foi observada diferen?a estat?stica entre a imunomarca??o da IL-17 e ROR?t com grada??o histol?gica de malignidade e com estadiamento cl?nico. Os achados dessa pesquisa sugerem um poss?vel papel antitumoral da IL-17 para os casos de l?bio. Os resultados da an?lise do ROR?t, possivelmente se devem ? ampla dualidade do papel pr?-tumoral e antitumoral das c?lulas Th17 e ? sua plasticidade que, na presen?a de diferentes citocinas expressas no microambiente tumoral, podem alterar seu fen?tipo