抑制腫瘤的基因（又稱抑癌基因）之表達失活，是導致癌變的重要機制之一。除了基因突變之外，越來越多研究證明抑癌基因的關閉轉錄，是由於抑癌基因啟動子區的CpG島甲基化所致。本論文的研究確定了兩個候選抑癌基因ADAMTS8和TUSC14，在多種腫瘤細胞株中經常因動子區的CpG島甲基化而下調或停止表達，這有別於它們在正常組織中廣泛表達的情況。沉默細胞株在脫氧核糖核酸甲基轉移酶的抑製劑5-氮-2'-脫氧胞苷(5-aza-2′-deoxycytidine; Aza) 或與組蛋白去乙酰化酶抑製劑曲古抑菌素A (trichostatin A, TSA)的去甲基化作用下，能恢復這兩個抑癌基因的表達，因而證明了啟動子甲基化是直接導致其表達下調及沉默的機制。 / 論文的第一部分，主要調查ADAMTS8啟動子區在原發腫瘤樣本被甲基化的比率，並研究其腫瘤抑制功能。含血小板凝血酶敏感蛋白基序的解整聯蛋白金屬蛋白酶 (ADAMTSs) ，在各種癌症中的表達異常已有報導。然而，它們在腫瘤的職能作用仍然模糊不清。本研究發現，異位表達ADAMTS8誘導細胞凋亡，因而顯著抑制腫瘤細胞克隆形成的能力,。這些都突顯其抑制腫瘤的功能。此外，作為分泌蛋白酶的ADAMTS8，能夠透過減少表皮生長因子受體(EGFR) 蛋白的磷酸化，抑制EGFR / MEK / ERK信號通路，並進一步破壞肌動蛋白應力纖維的組織，抑制腫瘤細胞的遷移性。 / 論文的第二部分，集中於研究一個未知功能的基因TUSC14，這基因的蛋白質編碼具有氨基末端蛋白質相互作用域 (BTB/POZ domain)及C₂H₂乙炔鋅指結構。TUSC14的異位表達能抑制腫瘤細胞克隆的形成，但這種抑制作用會在删除蛋白中的BTB/POZ或C₂H₂乙炔鋅指結構功能域後消失。因此證實了TUSC14蛋白同時需要BTB / POZ和C₂H₂乙炔鋅指結構兩個功能域來抑制腫瘤生長。此外，TUSC14具有抑制NF-kB轉錄的功能，其功能不但依賴於组蛋白去乙酰基酶(HDAC)，並且與c-MYC和cIAP-2等NF-κB靶基因下調表達相關。TUSC14的抑癌功能，包括抑制腫瘤生長與增加細胞凋亡，與其減少c-MYC及抗凋亡基因cIAP-2的表達，效果一致。進一步的分析發現，TUSC14與HDAC1和P65於蛋白質複雜免疫共沉澱實驗中，有物理相互作用。此外，染色質免疫沉澱實驗顯示TUSC14透過與c-MYC和cIAP-2的相互作用，抑制其基因啟動子區的轉錄功能。結果表明，TUSC14是通過招募HDAC至NF-κB靶基因的啟動子區這機制，來抑制NF-kB靶基因的轉錄，以達至抑制癌細胞生長和誘導癌細胞凋亡的效果。因此，TUSC14的沉默是破壞癌細胞中NF-kB信號通路負調控(negative regulation)的重要因素。 / 綜上所述，本研究鑒定了兩個在多種腫瘤細胞因表觀遺傳沉默效應而表達下調或沉默的抑癌基因ADAMTS8和TUSC14，並證實它們具有抑癌功能。 / Inactivation of tumor suppressor genes (TSGs) is one of the critical mechanisms leading to carcinogenesis. Apart from genetic mutations, a growing number of TSG has been shown to be silenced through promoter CpG methylation. In this thesis, we identified two candidate TSGs: ADAMTS8 and TUSC14 that are frequently downregulated or silenced in multiple carcinoma cell lines by promoter methylation while broadly expressed in normal tissues. Expression of these two genes was restored after treatment with DNA methyltransferase inhibitor 5-aza-2'-deoxycytidine (Aza) or in combination with a histone deacetylase inhibitor trichostatin A (TSA), suggesting promoter-methylation directly contributes to their silencing. / In the first part of the thesis, prevalence silencing of ADAMTS8 was detected in primary tumor samples. Expression of many disintegrins and metalloproteinases with thrombospondin motifs (ADAMTSs) was reported to be dysregulated in various cancers. However, their functional roles in tumorigenesis remain obscure. This study revealed that ectopic expression of ADAMTS8 markedly inhibits tumor cell clonogenicity by inducing apoptosis, underscoring its function as a tumor suppressor. Furthermore, ADAMTS8, as a secreted protease, inhibits EGFR/MEK/ERK signaling pathway by reducing their phosphorylation, further resulting in the disruption of actin stress fiber organization and suppression of tumor cell motility. / The second part of the thesis focused on a novel gene TUSC14 which encodes a protein with BTB/POZ domain and C₂H₂zinc-fingers. Ectopic expression of TUSC14 suppresses colony formation of cancer cells but this inhibitory effect is abolished with the deletion of BTB/POZ domain or C₂H₂ zinc-fingers. This suggested that both BTB/POZ domain and C₂H₂ zinc-fingers are required for inhibiting tumor cell clonogenecity. In addition, TUSC14 functions as a transcriptional repressor of NF-kB pathway that is dependent on HDAC. Suppression of NF-κB transcriptional activity by TUSC14 expression correlates with the downregulation of NF-κB target genes including c-MYC and cIAP-2. Reduction of c-MYC and anti-apoptotic cIAP-2 agrees well with the consequent growth suppression and enhanced apoptosis following the ectopic expression of TUSC14. Further analyses showed TUSC14 physically interacts with HDAC1 and p65 via co-immunoprecipitation assay. Preliminary ChIP assay showed that TUSC14 associates with gene promoters of c-MYC and cIAP-2 for their transcription repressions. These results revealed that TUSC14 represses NF-kB activity through recruiting HDAC to the NF-kB target genes; and transcription repression of NF-kB represents a mechanism for TUSC14 to mediate its growth inhibitory and apoptosis-inducing effects in cancer. Hence, silencing of TUSC14 contributes to the lost of negative regulation on NF-kB signaling in cancer. / In summary, this study demonstrated that ADAMTS8 and TUSC14 are functional tumor suppressors that are epigenetically silenced in multiple tumors. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Choi, Ching Gee. / Thesis (Ph.D.)--Chinese University of Hong Kong, 2012. / Includes bibliographical references (leaves 140-153). / Abstract also in Chinese. / Abstract --- p.i / Chinese abstract --- p.iv / AcknowledgEments --- p.vii / List of Figures --- p.ix / List of Tables --- p.xi / LIST OF ABBREVIATIONS --- p.xii / List of PUBLICATIONs --- p.xiv / Chapter CHAPTER 1 --- Introduction --- p.1 / Chapter 1.1 --- Overview of cancer epigenetics --- p.1 / Chapter 1.2 --- Epigenetic events --- p.2 / Chapter 1.2.1 --- DNA methylation --- p.2 / Chapter 1.2.2 --- Histone modifications --- p.5 / Chapter 1.2.3 --- The interdependence of DNA methylation and histone modifications --- p.8 / Chapter 1.3 --- Epigenetic alterations in cancer --- p.9 / Chapter 1.3.1 --- Genome-wide DNA hypomethylation --- p.9 / Chapter 1.3.2 --- CpG island promoter hypermethylation silencing of tumour suppressor genes in tumorigenesis --- p.10 / Chapter 1.3.3 --- Aberrations of histone modifications --- p.11 / Chapter 1.4 --- Causes for epigenetic deregulation in cancer --- p.14 / Chapter 1.5 --- The interplay of genetic and epigenetic aberration in cancer progression --- p.21 / Chapter 1.6 --- Epigenetic inactivation of tumor suppressor genes in cancer --- p.23 / Chapter 1.7 --- Clinical implications of epigenetic research --- p.27 / Chapter 1.7.1 --- Epigenetic modifications as biomarker for cancer diagnosis --- p.27 / Chapter 1.7.2 --- Targeting epigenetic modifications as therapeutics towards cancers --- p.29 / Chapter 1.8 --- Roles of ADAMTS proteins in cancer --- p.32 / Chapter 1.8.1 --- Introduction on ADAMTS metalloproteases --- p.32 / Chapter 1.8.2 --- Deregulation of ADAMTS protein in cancer --- p.34 / Chapter 1.9 --- Roles of BTB/POZ-ZF family of transcription factors in cancer --- p.36 / Chapter 1.9.1 --- Introduction on BTB/POZ-ZF Family --- p.36 / Chapter 1.9.2 --- BTB/POZ-ZF functions as transcription repressors --- p.37 / Chapter 1.9.3 --- Many BTB/POZ-ZF proteins are important players in tumorigenesis --- p.39 / Chapter 1.9.4 --- The role of BTB/POZ-ZF in tumor initiation and progression --- p.40 / Chapter CHAPTER 2 --- Aims of this study --- p.44 / Chapter CHAPTER 3 --- General Methodology --- p.46 / Chapter 3.1 --- Cell Culture --- p.46 / Chapter 3.1.1 --- Growth and maintenance of cells --- p.46 / Chapter 3.1.2 --- Mammalian cell transfection --- p.46 / Chapter 3.1.3 --- Drug and stress treatments --- p.47 / Chapter 3.2 --- DNA and RNA extraction --- p.47 / Chapter 3.3 --- Semi-quantitative RT-PCR and Real-time PCR --- p.48 / Chapter 3.4 --- CpG island and Transcription factor binding sites analysis --- p.49 / Chapter 3.5 --- Methylation-specific PCR (MSP) and Bisulfite genomic sequencing (BGS) --- p.49 / Chapter 3.6 --- Bacterial transformation and Plasmid extraction --- p.50 / Chapter 3.6.1 --- Heat-shock transformation --- p.50 / Chapter 3.6.2 --- Mini-scale preparation of plasmid DNA --- p.51 / Chapter 3.6.3 --- Preparation of endotoxin-free plasmids --- p.52 / Chapter 3.7 --- DNA cycle sequencing --- p.52 / Chapter 3.8 --- Indirect immunofluorescence for subcellular localization study --- p.54 / Chapter 3.9 --- Colony formation assay --- p.54 / Chapter 3.10 --- Cell cycle analysis --- p.55 / Chapter 3.11 --- Apoptosis assay --- p.56 / Chapter 3.12 --- Co-immunoprecipitation and Western blot --- p.56 / Chapter 3.13 --- Chromatin immunoprecipitation (ChIP) --- p.58 / Chapter 3.14 --- Dual Firefly and Renilla luciferase reporter gene assay --- p.59 / Chapter 3.15 --- Statistical analysis --- p.60 / Chapter CHAPTER 4: --- Characterization of the Tumor Suppressive Functions of ADAMTS8 --- p.61 / Chapter 4.1 --- Introduction --- p.61 / Chapter 4.2 --- Materials and Methods --- p.64 / Chapter 4.2.1 --- Tumor samples --- p.64 / Chapter 4.2.2 --- Expression of ADAMTS8 --- p.64 / Chapter 4.2.3 --- Immunofluorescence staining of ADAMTS8 --- p.64 / Chapter 4.2.4 --- Detection of secreted ADAMTS8 in culture medium --- p.65 / Chapter 4.2.5 --- Collection of conditioned medium and Western Blotting --- p.66 / Chapter 4.2.6 --- Wound healing assay --- p.66 / Chapter 4.3 --- Result and Discussion --- p.69 / Chapter 4.3.1 --- Frequent ADAMTS8 methylation in primary carcinomas --- p.69 / Chapter 4.3.2 --- ADAMTS8 is a secreted protease --- p.70 / Chapter 4.3.3 --- ADAMTS8 inhibits phosphorylation of pEGFR --- p.73 / Chapter 4.3.4 --- ADAMTS8 suppresses cell migration --- p.77 / Chapter 4.4 --- Summary --- p.81 / Chapter CHAPTER 5: --- Epigenetic Alterations of TUSC14 Gene in multiple carcinomas --- p.83 / Chapter 5.1 --- Introduction --- p.83 / Chapter 5.2 --- Materials and Methods --- p.84 / Chapter 5.2.1 --- Cell lines --- p.84 / Chapter 5.2.2 --- Normal and primary tumor tissues --- p.85 / Chapter 5.3 --- Results and Discussion --- p.86 / Chapter 5.3.1 --- Expression profiling of TUSC14 in normal tissues and tumor cell lines --- p.86 / Chapter 5.3.2 --- Frequent inactivation of TUSC14 by promoter CpG methylation --- p.90 / Chapter 5.3.3 --- Pharmacologic and genetic demethylation restores TUSC14 expression --- p.94 / Chapter 5.3.4 --- Frequent TUSC14 methylation in primary tumors --- p.96 / Chapter 5.4 --- Summary --- p.98 / Chapter CHAPTER 6 --- Characterization of the Tumor Suppressive Functions of TUSC14 --- p.99 / Chapter 6.1 --- Introduction --- p.91 / Chapter 6.2 --- Materials and Methods --- p.100 / Chapter 6.2.1 --- Gene cloning and plasmids construction of TUSC14 --- p.100 / Chapter 6.2.2 --- Drug and stress treatments of cells --- p.100 / Chapter 6.3 --- Results and Discussion --- p.102 / Chapter 6.3.1 --- TUSC14 localizes to nuclear speckles --- p.102 / Chapter 6.3.2 --- TUSC14 inhibits clonogenicity --- p.107 / Chapter 6.3.3 --- Expression of TUSC14 induces apoptosis in tumor cells --- p.110 / Chapter 6.3.4 --- TUSC14 alters cell cycle progression --- p.112 / Chapter 6.3.5 --- TUSC14 acts as a transcriptional repressor of multiple genes --- p.114 / Chapter 6.3.6 --- TUSC14 represses NF-кB activity through an HDAC-dependent mechanism --- p.118 / Chapter 6.3.7 --- The effect of TUSC14 on the expression of downstream targets of NF-κB Signaling --- p.120 / Chapter 6.3.8 --- TUSC14 co-immunoprecipitates with HDAC1 and p65 --- p.124 / Chapter 6.3.9 --- ChIP analysis of promoters of TUSC14-regulated genes --- p.127 / Chapter 6.4 --- Summary --- p.130 / Chapter CHAPTER 7 --- General Discussion --- p.133 / Chapter CHAPTER 8 --- Conclusions --- p.138 / References --- p.140
| Identifer | oai:union.ndltd.org:cuhk.edu.hk/oai:cuhk-dr:cuhk_328439 |
| Date | January 2012 |
| Contributors | Choi, Ching Gee., Chinese University of Hong Kong Graduate School. Division of Medical Sciences. |
| Source Sets | The Chinese University of Hong Kong |
| Language | English, Chinese |
| Detected Language | English |
| Type | Text, bibliography |
| Format | electronic resource, electronic resource, remote, 1 online resource (xiv, 153 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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