全球人類健康的頭號殺手--腫瘤目前仍是難以攻克的醫學難題。腫瘤的發生是一個復雜的過程，主要由促癌基因的異常增多或激活及抑癌基因（TSG）的缺失或功能喪失的累積效果導致。近年來基於非基因序列改變所致基因表達水平變化的表觀遺傳學的研究進展表明，啟動子區CpG島甲基化所致的表觀遺傳沉默是抑癌基因轉錄失活的重要機制。Ras和Wnt信號轉導通路在癌病的發生和發展過程中均起到重要的作用，因此針對該兩種信號通路的拮抗因子的表觀遺傳調控及功能學研究將為我們提供有研究及應用前景的候選抑癌基因。 / 作為一種重要的原癌基因，Ras家族基因具有致癌活性的點突變及其導致的過度激活的Ras信號通路被發現廣泛存在於大約30%的人類腫瘤中。然而在一些缺乏Ras基因突變的腫瘤類型中，持續激活的Ras信號通路仍然普遍存在並具有重要作用，昭示著除了Ras基因點突變以外的信號轉導異常激活的機制。與GTP的結合可激活Ras，而RasGAP家族蛋白可通過水解GTP達到使Ras失活的作用。通過采用微陣列比較基因組雜交(aCGH)的實驗手段我們發現6p21.3染色體區具有半接合子缺失， 並於此區域發現了候選抑癌基因RASA5。在以往的研究報道中，RASA5被命名為SynGAP且其功能研究僅限於神經系統。我們的研究發現不同於RasGAP家族的其它基因RASA2-4，RASA5廣泛表達於人類正常器官組織中，並特異性地在腫瘤細胞，特別是鼻咽癌(NPC)，食管鱗狀上皮細胞癌(ESCC)和乳腺癌這些具有野生型Ras基因但Ras信號通路仍被過度激活的細胞中被表觀遺傳沉默。RASA5的異位表達可有效促進腫瘤細胞的雕亡，抑制腫瘤細胞的生長、遷移及“幹性（stemness）“。同時，使用siRNA敲除內源性RASA5可以激發細胞的克隆形成及上皮-間質(EMT)轉化。RASA5的抑癌功能是通過調低Ras-GTP水平並進而抑制其下遊信號通路的活性實現的。過量表達具有致癌活性的點突變的Ras或RasGAP結構域缺失均可部分逆轉這種抑癌作用。此項研究首次證明了RASA5的抑癌功能。 / Wnt/Dvl/β-catenin信號轉導通路在人類腫瘤中存在廣泛的異常激活。我們發現DACT (Dpr/Frodo)家族成員TUSC-T2的表觀遺傳沉默是一種普遍存在於人類腫瘤中的現象。TUSC-T2編碼一種胞質蛋白，外源性表達TUSC-T2可促進腫瘤細胞雕亡並導致腫瘤細胞的克隆形成能力下降。TUSC-T2可與Dvl蛋白結合並下調其活化水平，從而保護GSK-3β蛋白不被Dvl蛋白抑制。GSK-3β可與Axin及APC蛋白形成蛋白質復合物，該復合物可捕捉並降解細胞內信號分子β-catenin。TUSC-T2的過量表達可以抑制β-catenin的激活及其向細胞核內的富集，並進一步阻止β-catenin在細胞核內與Lef/Tcf轉錄因子家族的作用及下遊特定原癌基因，例如c-Myc, CCND1及Fibronectin的表達。因此TUSC-T2具有抑制腫瘤細胞增殖、遷移及上皮-間質(EMT)轉化的作用。 / 綜上所述，我們的研究結果表明RASA5及TUSC-T2是具有抑癌功能的Ras或Wnt/Dvl/β-catenin信號轉導通路抑制因子，其表觀遺傳沉默導致的轉錄失活對於腫瘤的發生發展具有重要意義。同時，針對這兩種抑癌基因的進一步研究將為我們提供富有應用前景的腫瘤標記物。值得註意的是，RASA5課題的研究開創性地闡明了Ras信號通路的拮抗因子的表觀遺傳沉默是一種Ras信號轉導通路於腫瘤細胞中異常激活的新機制。 / Cancer is the top killer of the world, as well as the medical problem difficult to overcome. The conversion of a normal cell to a cancer cell is usually caused by upregulation of oncogenes and downregulation of tumor suppressor genes (TSGs). Epigenetic silencing has been proved to be important in TSGs inactivation, often through methylation of CpG-rich promoter regions. Ras and Wnt signaling pathways are both important for the tumorigenesis, epigenetic and functional studies of antagonists to Ras and Wnt signaling would provide us with candidate TSGs. / Ras is a well-known oncogene. Aberrant mutations of Ras genes occur in approximately 30% of human tumors, causing constitutively activated Ras signaling. However, in certain types of tumors with wild type Ras genes, abnormally activated Ras signaling is still a common and critical event, suggesting alternative mechanisms for Ras signaling hyperactivation. Ras is active when it is bound to GTP, while the hydrolysis of bound GTP and inactivation of Ras is catalyzed by Ras GTPase activating proteins (RasGAPs). Using 1-Mb array CGH (aCGH), we refined a small hemizygous deletion at the 6p21.3 chromosome region that contains a RasGAP family member gene RASA5, which used to be named as SynGAP and studied only in the neuron systems. We demonstrated that RASA5, rather than other RasGAP family members RASA2-4, is broadly expressed in human normal tissues while frequently epigenetically silenced in multiple tumors, especially in certain tumor types such as nasopharyngeal (NPC), esophageal (ESCC) and breast carcinomas (BrCa) with wild-type Ras while Ras cascade is still constitutively active. Ectopic expression of RASA5 led to apoptosis, growth and migration inhibition, as well as ‘stemness’ repression of tumor cells. Meanwhile, knockdown of RASA5 by siRNA promoted the tumor cell colony formation as well as epithelial-mesenchymal transition (EMT). The tumor-suppressive function of RASA5 was exerted through downregulating Ras-GTP level and further inactivating Ras signaling. Such an inhibitory effect could be partially abrogated in the presence of mutated, activated Ras or by deletion of the RasGAP domain. For the first time, our study refined the role of RASA5 as a tumor suppressor. / Wnt/DVL/β-catenin signaling pathway is aberrantly activated in a wide range of human cancers. We identified a DACT (Dpr/Frodo) family member TUSC-T2 as an epigenetically downregulated gene in human tumors. TUSC-T2 encodes a punctate cytoplasmic protein. Ectopic expression of TUSC-T2 dramatically inhibited tumor cell colony formation in silenced tumor cell lines, mainly through inducing apoptosis. TUSC-T2 interacts and downregulates Dishevelled (Dvl) protein, thus protecting glycogen synthase kinase 3β (GSK-3β) from inactivation by Wnt/Dvl and allowing GSK-3β to form a complex with Axin and APC to promote the phosphorylation and proteasomal degradation of β-catenin. Overexpression of TUSC-T2 disrupted β-catenin activation and accumulation in nuclei, thus preventing its binding to transcription factors of the Lef/Tcf family. This caused the downregulation of β-catenin target oncogenes such as c-Myc, CCND1 and Fibronectin as well as the inhibition of tumor cell proliferation and migration. We also observed that TUSC-T2 could inhibit tumor cell EMT. / Taken together, our data demonstrate that RASA5 and TUSC-T2 are functional tumor suppressors epigenetically silenced in multiple tumors through acting as negative regulators of the Ras or Wnt/Dvl/β-catenin cancer pathways, and could be developed as promising biomarkers for human tumors. Of note, our study reveals that epigenetic silencing of the Ras antagonist represents a new mechanism responsible for Ras aberrant activation in cancers with wild-type Ras. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Fan, Yichao. / Thesis (Ph.D.)--Chinese University of Hong Kong, 2012. / Includes bibliographical references (leaves 184-216). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Abstract also in Chinese. / Acknowledgements --- p.i / List of abbreviations --- p.ii-iii / List of tables --- p.iv / List of Figures --- p.v-vii / List of Publications --- p.viii-ix / Abstract in English --- p.x-xii / Abstract in Chinese --- p.xiii-xiv / Table of Contents --- p.xv / Chapter Chapter 1 --- Introduction and Literature Review --- p.1 / Chapter 1.1 --- Cancer epigenetics --- p.4 / Chapter 1.1.1 --- Epigenetic modifications --- p.5 / Chapter 1.1.1.1 --- DNA Methylation --- p.5 / Chapter 1.1.1.2 --- Histone modifications --- p.10 / Chapter 1.1.1.3 --- RNA interference --- p.14 / Chapter 1.1.1.4 --- Nucleosome positioning --- p.15 / Chapter 1.1.2 --- Epigenetic alteration induced Tumor suppressor genes (TSGs) silencing during carcinogenesis --- p.17 / Chapter 1.2 --- Epigenetic alterations in cancer pathways --- p.23 / Chapter 1.2.1 --- Brief introduction of cancer pathways --- p.23 / Chapter 1.2.2 --- Ras pathway --- p.25 / Chapter 1.2.2.1 --- Ras pathway and carcinogenesis --- p.25 / Chapter 1.2.2.2 --- Epigenetic regulation of RasGAP proteins in carcinogenesis --- p.28 / Chapter 1.2.2.3 --- Epigenetic silencing of other negative regulators of Ras signaling --- p.30 / RAS association domain family (RASSF) proteins --- p.30 / PTEN --- p.32 / Sprouty (SPRY) proteins --- p.33 / Chapter 1.2.2.4 --- Hypomethylation induced Ras oncogenes activation --- p.35 / Chapter 1.2.2.5 --- Ras mediates epigenetic regulation through feedback loop --- p.36 / Chapter 1.2.3 --- Wnt pathway --- p.43 / Chapter 1.2.3.1 --- Wnt signaling pathway and carcinogenesis --- p.43 / Chapter 1.2.3.2 --- Epigenetic silencing of negative regulators of Wnt signaling --- p.45 / Chapter 1.2.3.3 --- DACT family proteins and carcinogenesis --- p.48 / Chapter 1.3 --- Application of tumor specific epigenetic alterations as tumor biomarkers and therapeutic targets --- p.49 / Chapter 1.3.1 --- The potential and advantage of tumor specific epigenetic alterations used as tumor biomarkers and therapeutic targets --- p.49 / Chapter 1.3.2 --- Epigenetic-disrupted regulators of Ras signaling as tumor biomarkers and therapeutic targets --- p.50 / Chapter 1.3.3 --- Epigenetic-disrupted regulators of Wnt signaling as tumor biomarkers and therapeutic targets --- p.52 / Chapter Chapter 2 --- Aims of this study --- p.54 / Chapter 2.1 --- To identify epigenetically silenced candidate TSGs as antagonists to Ras or Wnt signaling --- p.55 / Chapter 2.2 --- To elucidate the functional of candidate TSGs --- p.56 / Chapter Chapter 3 --- Materials and Methods --- p.57 / Chapter 3.1 --- Cell lines, tumor samples and routine cell line maintenance --- p.57 / Chapter 3.2 --- Drug and stress treatments --- p.59 / Chapter 3.3 --- DNA and RNA extraction --- p.59 / Chapter 3.4 --- Semi-quantitative RT-PCR and Real time PCR --- p.60 / Chapter 3.5 --- Direct sequencing of PCR products --- p.67 / Chapter 3.6 --- CpG island analysis --- p.67 / Chapter 3.7 --- Bisulfite treatment --- p.67 / Chapter 3.8 --- Methylation-specific PCR (MSP) and bisulfite genomic sequencing --- p.68 / Chapter 3.9 --- Plasmid extraction --- p.69 / Chapter 3.9.1 --- Bacteria culture --- p.69 / Chapter 3.9.2 --- Mini-scale preparation of plasmid DNA --- p.70 / Chapter 3.9.3 --- Large-scale endotoxin-free plasmids extraction --- p.71 / Chapter 3.10 --- Construction of expression plasmids --- p.71 / Chapter 3.10.1 --- Gene cloning and plasmids construction of RASA5 --- p.71 / Chapter 3.10.2 --- Gene cloning and plasmids construction of TUSC-T2 --- p.74 / Chapter 3.11 --- Immunofluorescence Staining --- p.74 / Chapter 3.12 --- Colony formation assay --- p.76 / Chapter 3.13 --- Apoptosis assay --- p.77 / Chapter 3.14 --- Luciferase reporter assay --- p.78 / Chapter 3.15 --- Protein preparation and Western blot --- p.79 / Chapter 3.16 --- Ras Activity Assay --- p.80 / Chapter 3.17 --- Wound healing assay --- p.81 / Chapter 3.18 --- Matrigel invasion assay --- p.81 / Chapter 3.19 --- RNA Interference --- p.81 / Chapter 3.20 --- Statistical analysis --- p.82 / Chapter Chapter 4: --- Epigenetic disruption of Ras signaling through silencing of a Ras GTPase-activating protein RASA5 in human cancers --- p.83 / Chapter 4.1 --- Identification of RASA5 as a downregulated gene residing in the 6p21.3 deletion region --- p.86 / Chapter 4.2 --- RASA5 is widely expressed in human normal tissues but downregulated in tumor cell lines --- p.91 / Chapter 4.3 --- The tumor-specific downregulation pattern of RASA5 is unique in the RASA family genes --- p.95 / Chapter 4.4 --- RASA5 promoter CpG methylation resulted in its transcription inactivation --- p.96 / Chapter 4.5 --- Frequent methylation of RASA5 promoter in multiple primary tumors --- p.101 / Chapter 4.6 --- Cloning and characterization of human RASA5 --- p.104 / Chapter 4.7 --- RASA5 inhibits tumor cell clonogenicity through inducing apoptosis --- p.108 / Chapter 4.8 --- RasGAP domain is required for the tumor suppressive function of RASA5 --- p.111 / Chapter 4.9 --- Certain cancer types harbor wild type Ras but active Ras signaling, with RASA5 epigenetically silenced --- p.114 / Chapter 4.10 --- RASA5 antagonizes Ras signaling pathway --- p.117 / Chapter 4.10.1 --- RASA5 represses Ras signaling through downregulating Ras-GTP level --- p.117 / Chapter 4.10.2 --- Oncogenic mutant form of Ras abrogated colony formation inhibitory effect of RASA5 on tumor cells --- p.120 / Chapter 4.10.3 --- Knockdown of RASA5 promoted the tumor cell colony formation and Ras signaling activation --- p.122 / Chapter 4.10.4 --- RASA5 inhibits ERK1/2 nuclei translocation and activation --- p.123 / Chapter 4.10.5 --- RASA5 negatively regulates Ras target gene expression --- p.125 / Chapter 4.11 --- RASA5 inhibits tumor cell migration and invasion through the Ras/Rac/cofilin signaling --- p.127 / Chapter 4.12 --- RASA5 suppresses tumor cell epithelial-mesenchymal transition (EMT) and stemness --- p.133 / Chapter 4.13 --- RASA5 appears in the cellcell interaction region nanotubes --- p.139 / Chapter 4.14 --- Discussion --- p.141 / Chapter Chapter 5: --- The Wnt/Dvl signaling antagonist TUSC-T2 is a pro-apoptotic tumor suppressor epigenetically silenced in tumors and inhibits tumor cell proliferation and migration --- p.150 / Chapter 5.1 --- Expression of TUSC-T2 is downregulated in human tumors --- p.150 / Chapter 5.2 --- TUSC-T2 promoter methylation results in its transcriptional inactivation --- p.151 / Chapter 5.3 --- Cloning and characterization of TUSC-T2 --- p.155 / Chapter 5.4 --- TUSC-T2 inhibits tumor cell clonogenicity through inducing apoptosis --- p.157 / Chapter 5.5 --- TUSC-T2 inhibits Wnt/Dvl/β-catenin pathway --- p.161 / Chapter 5.6 --- TUSC-T2 suppresses cell migration and EMT through upregulating E-cadherin --- p.165 / Chapter 5.7 --- Discussion --- p.171 / Chapter Chapter 6: --- Conclusions --- p.176 / Chapter 6.1. --- RasGAP family member RASA5 is epigenetically silenced in human cancers, acting as a tumor suppressor through negatively regulating Ras signaling --- p.177 / Chapter 6.2. --- DACT family member TUSC-T2 functions as a candidate TSG silenced by promoter methylation and inhibits Wnt/Dvl/β-catenin pathway --- p.178 / Chapter Chapter 7: --- Future Studies --- p.181 / Chapter 7.1. --- Further functional study of RASA5 and TUSC-T2 --- p.181 / Chapter 7.2. --- Clinical application of epigenetic silenced candidate TSGs --- p.182 / Chapter 7.3. --- Further screening of candidate TSGs as antagonists to cancer pathways --- p.183 / Reference list --- p.184
| Identifer | oai:union.ndltd.org:cuhk.edu.hk/oai:cuhk-dr:cuhk_328120 |
| Date | January 2012 |
| Contributors | Fan, Yichao., 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 (xviii, 216 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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