運用miRNA微陣列手段，我們篩選到一批在結直腸腫瘤內高表達和低表達的miRNA。其中miR-133a是在腫瘤中最顯著降低的miRNA之一， 但其在腫瘤的發生發展過程中的作用尚未可知，因此我們選擇miR-133a最為研究目標，論證其的生物學功能，分子機理，及其在結直腸癌中的靶分子。 / 我們首先在較大規模樣本中驗證miR-133a的低表達。定量PCR結果顯示，對比正常的癌旁組織，miR-133a在94例結直腸癌組織中顯著低表達（p < 0.001）。我們進一步分析miR-133a在9種常用的腫瘤細胞系內的表達情況。對比正常的直腸組織，miR-133a在9種常用的結直腸細胞系內的表達均明顯下降。在腫瘤發生發展過程中，癌細胞趨向於降低具有抑癌功能的基因的表達。因此我們推測miR-133a是一個潛在的腫瘤抑制miRNA。 / 為了論證我們的假設，我們首先選取miR-133a低表達的結直腸癌細胞系HCT116和LoVo最為研究模型，將miR-133a在這兩種細胞系內過表達。升高的miR-133a可以抑制腫瘤細胞生長（p < 0.01和p < 0.05），抑制腫瘤克隆集落形成（p < 0.01）。我們進一步發現過表達miR-133a可以抑制裸鼠體內腫瘤的生長（p < 0.05）。細胞週期分析顯示miR-133a抑制細胞生長的能力表現為誘導腫瘤細胞週期阻滯於G0/G1期。細胞週期特異性CDK抑制蛋白p21和p27對於細胞週期調節十分關鍵。基於此項觀察，我們進一步分析了p21和p27的表達。Western-blot實驗證實過表達miR-133a可以促進HCT116和LoVo細胞內p21和p27的蛋白上調。但miR-133a在p53突變型的HT29過表達後並沒有引起p21的變化。通過對比p53野生型和突變性細胞系，我們發現p53對於miR-133a誘導p21是十分關鍵的。為了證明miR-133a可以引起p53的活性，我們通過啟動子螢光報告實驗證實，miR-133a不僅可以促進p53結合DNA 的能力，而且可以引起p21啟動子的活性。其次，我們發現在p53野生型細胞株HCT116中，轉染si-p53可以拮抗miR-133a誘導p21。我們通過蛋白降解實驗發現，miR-133a可以延長p53蛋白的半衰期。 / 基於以上實驗事實，我們推測miR-133a誘導p21是通過穩定p53蛋白來實現的。通過數個miRNA靶基因預測軟體，我們判斷RFFL可能是miR-133a發揮效力的直接靶基因。RFFL是E3連接酶，負責非磷酸化p53和磷酸化p53的降解。在RFFL的3側非翻譯區有一個進化保守的miR-133a識別序列。我們克隆此段序列到螢光素酶基因的3側非翻譯區，並進行雙螢光素酶報告基因檢測。測試發現miR-133a可以直接結合到RFFL的此段序列上，並抑制前段基因的翻譯。體外實驗證實，過表達miR-133a可以減少HCT116和HT29細胞內的RFFL蛋白，但並不影響其mRNA。過表達RFFL可以降低p53的表達，反之降低RFFL的蛋白表達，可以提高p53和p21蛋白。上述實驗均有助於證實miR-133a是通過抑制RFFL來提高p21的表達，進而抑制細胞週期。 / 我們也發現miR-133a具有增敏抗癌藥物的效力。單獨轉染miR-133a並不能顯著引發細胞凋亡，而聯合使用miR-133a及抗癌藥物doxorubicin，或者Oxaliplatin都可以顯著增強細胞的早期凋亡。 / 基於上述實驗結果，我們證實miR-133a是一個抑制腫瘤生長的miRNA，其機制可能是通過抑制RFFL蛋白的表達，並啟動p53/p21信號通路引起的。我們的實驗說明miR-133a可以對抗腫瘤藥物起到增敏的作用。 / We found that miR-133a was significantly down-regulated in colorectal cancer (CRC) tissues using miRNA array. However, the role of miR-133a in CRC is largely unknown. We sought to clarify its biologic function, molecular basis, and target gene in CRC. The down-regulation of miR-133a was verified in 94 primary CRC tumours compared with the matched adjacent normal tissues (p < 0.001) and in 9 human colon cancer cell lines. Ectopic expression of miR-133a in colon cancer cell lines (HCT116 and LoVo) significantly suppressed cell growth as evidenced by cell viability assay (p < 0.01 and p < 0.05), and colony formation assay (p < 0.01). Cell cycle analysis revealed that miR-133a caused an increased proportion of cells arrested at G0/G1 phase in HCT116 and LoVo, concomitant with the up-regulation of key G1 phase regulators CDK inhibitors p21 and p27. Promoter-luciferase activity assays indicated that miR-133a markedly increased p53 binding activity and induced p21 transcription. We further revealed that miR-133a decelerated p53 degradation, suggesting that miR-133a was an important positive modulator of the p53/p21 pathway. In silico search showed that the 3’UTR of ring finger and FYVE-like domain containing E3 ubiquitin protein ligase (RFFL), an E3 ubiquitin protein ligase targeting p53 for degradation, contains an evolutionarily conserved miR-133a binding site. Co-transfection with miR-133a repressed RFFL-3’UTR reporter activity for up to 53% (p < 0.01) and remarkably reduced RFFL protein level, indicating that miR-133a directly bound to RFFL mRNA and inhibited RFFL translation. Moreover, miR-133a sensitized colon cancer cells to chemotherapeutic agents (doxorubicin and oxaliplatin) by enhancing apoptosis (p < 0.01) and inhibiting cell proliferation (p < 0.001), adding further weight to the potential significance of miR-133a as a tumour suppressor in inhibiting CRC. In conclusion, miR-133a serves as a functional tumour suppressor in CRC through direct inhibition of the oncogenic RFFL and induction of the p53/p21 pathway. / 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. / Dong, Yujuan. / Thesis (Ph.D.)--Chinese University of Hong Kong, 2012. / Includes bibliographical references (leaves 139-152). / 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 / ACKNOWLEDGMENTS --- p.VI / LIST OF FIGURES --- p.VII / LIST OF TABLES --- p.IX / ABBREVIATIONS --- p.X / TABLES OF CONTENT --- p.XIII / Chapter CHAPTER ONE --- INTRODUCTION --- p.1 / Chapter 1.1 --- Colorectal cancer --- p.1 / Chapter 1.1.1 --- Epidemiology --- p.1 / Chapter 1.1.2 --- Etiology --- p.4 / Chapter 1.1.3 --- CRC prevention, screening and therapy --- p.7 / Chapter 1.2 --- microRNA (miRNA) --- p.13 / Chapter 1.2.1 --- Biogenesis of miRNA --- p.13 / Chapter 1.2.2 --- Diagnostic value of miRNAs in CRC --- p.16 / Chapter 1.2.3 --- Prognostic value of miRNAs in CRC --- p.25 / Chapter 1.2.4 --- Predictive value of miRNAs for treatment response in CRC --- p.28 / Chapter 1.2.5 --- miRNA-related single-nucleotide polymorphisms and CRC --- p.29 / Chapter 1.2.6 --- miRNAs and their function in CRC genesis --- p.33 / Chapter 1.2.7 --- Future perspective of miRNAs in CRC --- p.41 / Chapter 1.3 --- Hypothesis and objectives --- p.41 / Chapter CHAPER TWO --- METHODOLGY --- p.43 / Chapter 2.1 --- Cell cultures --- p.43 / Chapter 2.2 --- Patients and clinical specimens --- p.43 / Chapter 2.3 --- miRNA extraction from tissue and cell --- p.46 / Chapter 2.4 --- Micro-dissection and RNA extraction from paraffin sections --- p.47 / Chapter 2.5 --- Real-time quantitative PCR for miRNA microarray --- p.48 / Chapter 2.6 --- miRNA expression analysis --- p.48 / Chapter 2.7 --- mRNA expression analysis --- p.49 / Chapter 2.8 --- RNA interference --- p.52 / Chapter 2.9 --- Colony formation assay --- p.52 / Chapter 2.10 --- MTT cell viability assay --- p.53 / Chapter 2.11 --- Flow cytometry for cell cycle analysis --- p.53 / Chapter 2.12 --- Flow cytometry for cell apoptosis analysis --- p.54 / Chapter 2.13 --- Protein degradation assay --- p.54 / Chapter 2.14 --- Western blot analysis --- p.55 / Chapter 2.15 --- Immunofluorescence staining --- p.56 / Chapter 2.16 --- Plasmids construction --- p.58 / Chapter 2.16.1 --- pRFFL plasmid --- p.58 / Chapter 2.16.2 --- pMIR-RFFL-3’UTR and pMIR-RFFLmut-3’UTR --- p.58 / Chapter 2.17 --- Construction of stable cell lines --- p.59 / Chapter 2.18 --- Dual-luciferase reporter assay for p53 signaling pathway --- p.61 / Chapter 2.19 --- Dual-luciferase reporter assay for RFFL 3’UTR and miR-133a binding activity --- p.62 / Chapter 2.20 --- 5-Aza-2'-deoxycytidine (5-Aza-dC) treatment --- p.62 / Chapter 2.21 --- Tumour xenografts in nude mice model (miRNA intratumoural injection model) --- p.63 / Chapter 2.22 --- Tumour xenografts in nude mice model (miRNA stable cell line subcutaneous injection) --- p.64 / Chapter 2.23 --- Statistical analysis --- p.64 / Chapter CHAPTER THREE --- RESULTS --- p.66 / Chapter 3.1 --- Identification of differentially expressed miRNAs in CRC --- p.66 / Chapter 3.2 --- miR-133a is down-regulated in primary human CRC and colon cancer cell lines --- p.69 / Chapter 3.3 --- Ectopic expression of miR-133a inhibits tumourigenic properties of CRC cells --- p.75 / Chapter 3.3.1 --- miR-133a suppresses cell viability and colony formation --- p.75 / Chapter 3.3.2 --- miR-133a inhibits tumour growth in nude mice --- p.78 / Chapter 3.3.3 --- miR-133a suppresses cell cycle progression --- p.81 / Chapter 3.4 --- G0/G1 phase arrest by miR-133a is mediated through up-regulation of CDKN1A and CDKN1B --- p.83 / Chapter 3.5 --- miR-133a activates p53/p21 pathway through stabilization of p53 protein --- p.88 / Chapter 3.5.1 --- miR-133a induces p21 expression in a p53 wild-type cells --- p.88 / Chapter 3.5.2 --- miR-133a induces p21 promoter transcription activity and p53 binding activity --- p.90 / Chapter 3.5.3 --- Silence of p53 abolished miR-133a induced p21 --- p.92 / Chapter 3.5.4 --- miR-133a increases p53 activity through increase of p53 protein stability --- p.95 / Chapter 3.5.5 --- miR-133a has no effect on c-Myc level --- p.98 / Chapter 3.6 --- miR-133a increases p53 protein level by directly down-regulating RFFL --- p.100 / Chapter 3.7 --- Knock-down of RFFL inhibits cancer cell growth --- p.105 / Chapter 3.8 --- miR-133a sensitized CRC cells to chemotherapeutic drugs treatment --- p.110 / Chapter 3.9 --- Pharmacological demethylation restores miR-133a expression in CRC cells --- p.117 / Chapter 3.10 --- Association between miR-133a expression in tumour and clinicopathological characteristics of CRC patients --- p.119 / Chapter 3.11 --- Validation of other dysregulated miRNAs in CRC --- p.123 / Chapter CHAPTER FOUR --- DISCUSSION --- p.128 / Chapter 4.1 --- Biological role of miR-133a as a tumour suppressor --- p.128 / Chapter 4.2 --- p53/p21 pathway is a critical mediator of miR-133a in CRC --- p.129 / Chapter 4.3 --- Functional significance of RFFL in miR-133a induced p53/p21 signaling --- p.131 / Chapter 4.4 --- Clinical potential of miR-133a in CRC --- p.133 / Chapter 4.5 --- Other dysregulated miRNA --- p.136 / Chapter 4.6 --- Limitations and improvements of the study --- p.136 / Chapter 4.7 --- Conclusions --- p.137 / REFERENCE --- p.139 / PUBLICATIONS --- p.153

Xiong, Lei. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2013. / Includes bibliographical references (leaves 79-97). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Abstracts also in Chinese.

MicroRNAs play a key role in post-transcriptional gene regulation. They regulate target gene expression with mRNA degradation or translation repression. Each miRNA is estimated to regulate dozens of genes in human, and dysregulation of miRNA leads to various diseases, such as cancer, heart disease and depression. Therefore, it is critical to understand the mechanism of miRNA biogenesis and targeting. This work integrated gene and miRNA expression profile from various cancer projects to screen for potential miRNA biogenesis regulators and activity modulators. In this analysis, we identified several genes that regulate miRNA pathway and found their association with tumor progression and clinical outcome.

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

Lee, Kwan Yeung. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2007. / Includes bibliographical references (leaves 103-113). / Abstracts in English and Chinese. / Abstract --- p.i / Acknowledgements --- p.v / Table of Content --- p.vi / List of Figures --- p.xi / List of Tables --- p.xiii / List of Abbreviations --- p.xiv / List of papers published during the study --- p.xvi / Chapter Chapter 1 --- Introduction and Aim of Study --- p.1 / Chapter 1.1 --- General Introduction --- p.1 / Chapter 1.2 --- Project objective and potential significances --- p.5 / Chapter Chapter 2 --- Literatures Review --- p.6 / Chapter 2.1 --- Cancer genetics and Tumor suppressor genes --- p.6 / Chapter 2.2 --- Epigenetic --- p.7 / Chapter 2.2.1 --- DNA methylation and promoter CpG island --- p.8 / Chapter 2.2.2 --- Establishment and maintenance of DNA methylation --- p.9 / Chapter 2.2.3 --- Transcriptional silencing by DNA hypermethylation --- p.9 / Chapter 2.3 --- Cancer epigenetic --- p.11 / Chapter 2.3.1 --- Hypomethylation of the cancer genome --- p.12 / Chapter 2.3.2 --- Hypermethylation in cancers --- p.12 / Chapter 2.3.3 --- Clinical relevance of cancer epigenetic --- p.13 / Chapter 2.4 --- Nasopharyngeal carcinoma --- p.14 / Chapter 2.4.1 --- NPC genetic and epigenetic --- p.15 / Chapter 2.5 --- 12p as a putative tumor suppressor locus --- p.16 / Chapter 2.5.1 --- Hematological malignancies associated with 12p loss --- p.17 / Chapter 2.5.2 --- Prostate cancer associated with 12p loss --- p.20 / Chapter 2.5.3 --- Lung cancer associated with 12p loss --- p.22 / Chapter 2.5.4 --- 12p deletion in other cancers --- p.23 / Chapter 2.6 --- 16q as a tumor suppressor locus --- p.24 / Chapter 2.6.1 --- Breast cancer and 16q --- p.25 / Chapter 2.6.2 --- Loss of 16q and prostate cancer --- p.26 / Chapter 2.6.3 --- Loss of 16q and hepatocellular carcinoma --- p.28 / Chapter 2.6.4 --- 16q deletion associated with other cancers --- p.29 / Chapter Chapter 3 --- Materials and Methods --- p.30 / Chapter 3.1 --- Cell lines and tissue samples --- p.30 / Chapter 3.1.1 --- Cell lines --- p.30 / Chapter 3.1.2 --- Maintenance of cell lines --- p.31 / Chapter 3.1.3 --- Drugs treatment of cell lines --- p.31 / Chapter 3.1.4 --- Normal tissues --- p.32 / Chapter 3.1.5 --- Total RNA extraction --- p.32 / Chapter 3.1.6 --- Genomic DNA extraction --- p.32 / Chapter 3.2 --- General techniques --- p.33 / Chapter 3.2.2 --- TA cloning and blunt end cloning of PCR product --- p.33 / Chapter 3.2.3 --- Transformation of cloning products to E. coli competent cells --- p.34 / Chapter 3.2.4 --- Preparation of plasmid DNA --- p.34 / Chapter 3.2.4.1 --- Mini-prep plasmid DNA extraction --- p.34 / Chapter 3.2.4.2 --- Midi-prep of plasmid DNA --- p.35 / Chapter 3.2.5 --- Measurement of DNA or RNA concentrations --- p.36 / Chapter 3.2.6 --- DNA sequencing of plasmid DNA and PCR products --- p.36 / Chapter 3.3 --- Preparation of reagents and medium --- p.37 / Chapter 3.4 --- Semi-quantitative Reverse-Transcription (RT) PCR expression analysis --- p.38 / Chapter 3.4.1 --- Reverse transcription reaction --- p.38 / Chapter 3.4.2 --- Semi-quantitative RT-PCR --- p.39 / Chapter 3.4.2.1 --- Primers design --- p.39 / Chapter 3.4.2.2 --- PCR reaction --- p.39 / Chapter 3.5 --- Methylation analysis of candidate genes --- p.40 / Chapter 3.5.1 --- Bisulfite treatment of genomic DNA --- p.41 / Chapter 3.5.2 --- Methylation-specific PCR (MSP) --- p.42 / Chapter 3.5.2.1 --- Bioinformatics prediction of CpG island --- p.42 / Chapter 3.5.2.2 --- Primers design --- p.42 / Chapter 3.5.2.3 --- PCR reaction --- p.42 / Chapter 3.5.3 --- Bisulfite Genomic Sequencing (BGS) --- p.43 / Chapter 3.5.3.1 --- Primers design --- p.43 / Chapter 3.5.3.2 --- PCR reaction --- p.44 / Chapter 3.6 --- Construction of expression vectors of candidate genes --- p.44 / Chapter 3.6.1 --- Construction of IRF8 expression vector --- p.44 / Chapter 3.6.2 --- Construction of PTPRO expression vector --- p.44 / Chapter 3.6.2.1 --- Experimental design --- p.44 / Chapter 3.6.2.2 --- PCR and cloning of PCR products --- p.46 / Chapter 3.6.2.3 --- Restriction digestion of cloning vectors and expression vector --- p.48 / Chapter 3.6.2.4 --- Ligation of cloning fragments --- p.48 / Chapter 3.7 --- Colony formation assay on monolayer culture --- p.48 / Chapter 3.8 --- Statistical analysis --- p.49 / Chapter Chapter 4 --- Identification of candidate TSGs in deleted regions --- p.50 / Chapter 4.1 --- Research plan --- p.50 / Chapter 4.2 --- Results --- p.50 / Chapter 4.2.1 --- Mapping of the deleted B AC clones on their chromosomal locations --- p.50 / Chapter 4.2.2 --- Identification of down-regulated genes in NPC by semi-quantitative RT-PCR analysis --- p.51 / Chapter 4.3 --- Discussion --- p.55 / Chapter Chapter 5 --- Tumor suppressor function studies of candidate TSGs --- p.60 / Chapter 5.1 --- Research plan --- p.60 / Chapter 5.2. --- IRF8 is the 16q candidate TSG --- p.60 / Chapter 5.2.1 --- Frequent silencing of IRF8 mRNA expression in multiple carcinomas --- p.60 / Chapter 5.2.2 --- Methylation status of IRF8 promoter region correlated with its transcriptional silencing --- p.62 / Chapter 5.2.3 --- Restoration of IRF8 expression by pharmacological and genetic demethylation --- p.65 / Chapter 5.2.4 --- IRF8 inhibited the anchorage dependent growth of tumor cells on monolayer culture --- p.67 / Chapter 5.2.5 --- Discussion --- p.68 / Chapter 5.3 --- PTPRO is the down-regulated target at 12pl3.2-12.3 tumor suppressor locus --- p.73 / Chapter 5.3.1 --- Frequent silencing of PTPRO in multiple carcinoma cell lines --- p.73 / Chapter 5.3.2 --- Frequent methylation of PTPRO promoter CpG island in multiple carcinoma cell lines correlated with its reduced expression --- p.74 / Chapter 5.3.3 --- Re-expression of PTPRO by pharmacological and genetic demethylation --- p.77 / Chapter 5.3.4 --- PTPRO inhibited the growth of tumor cells in vitro --- p.79 / Chapter 5.3.5 --- Discussion --- p.81 / Chapter 5.4 --- RERG is another candidate TSG in 12pl3.2 - 12.3 region --- p.87 / Chapter 5.4.1 --- Down-regulation of RERG mRNA expression in carcinoma cell line --- p.87 / Chapter 5.4.2 --- Hypermethylation of RERG promoter is a frequent event in multiple carcinomas --- p.88 / Chapter 5.4.3 --- Re-expression of RERG mRNA following pharmacological and genetic demethylation --- p.90 / Chapter 5.4.4 --- Discussion --- p.92 / Chapter Chapter 6 --- General discussion --- p.96 / Chapter Chapter 7 --- Summary --- p.101 / Reference --- p.103

Ng, Ka Man. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2007. / Includes bibliographical references (leaves 103-113). / Abstracts in English and Chinese. / Acknowledgements --- p.i / List of abbreviations --- p.ii / List of Tables --- p.iv / List of Figures --- p.v / List of Publications --- p.viii / Abstract in English --- p.ix / Abstract in Chinese --- p.xi / Table of Contents --- p.xiii / Chapter Chapter 1 --- Literature Review --- p.1 / Chapter 1.1 --- Tumor suppressor genes (TSGs) and the modes of TSG inactivation during carcinogenesis --- p.1 / Chapter 1.2 --- Epigenetic modifications --- p.3 / Chapter 1.2.1 --- DNA methylation --- p.4 / Chapter 1.2.1a --- Establishment of DNA methylation patterns and DNA methyltransferases --- p.5 / Chapter 1.2.1b --- DNA hypermethylation and carcinogenesis --- p.6 / Chapter 1.2.1c --- Mechanism for gene silencing by CpG methylation --- p.6 / Chapter 1.2.1d --- DNA hypomethylation and carcinogenesis --- p.10 / Chapter 1.2.1e --- Loss of imprinting and carcinogenesis --- p.11 / Chapter 1.2.1f --- Potential factors leading to aberrant methylation patterns in cancers --- p.12 / Chapter 1.2.2 --- Deregulation of histone modifications and carcinogenesis --- p.14 / Chapter 1.2.3 --- Interplay between chromatin modifications and DNA methylation --- p.15 / Chapter 1.3 --- Identification of tumor suppressor genes (TSGs) --- p.17 / Chapter 1.4 --- Nasopharyngeal carcinoma as a cancer model of the current project --- p.18 / Chapter 1.5 --- Genetic and epigenetic changes in NPC --- p.19 / Chapter 1.6 --- Involvement of 5qll-ql2 and 14q32 in carcinogenesis --- p.22 / Chapter 1.6.1 --- Chromosome 5ql l-ql2 and carcinogenesis --- p.22 / Chapter 1.6.2 --- Chromosome 14q32 and carcinogenesis --- p.24 / Chapter 1.7 --- Clinical implications of epigenetics in cancers --- p.27 / Chapter Chapter 2 --- Aims of study and Research plan --- p.31 / Chapter Chapter 3 --- Materials and Methods --- p.34 / Chapter 3.1 --- Cell lines and Normal Tissues --- p.35 / Chapter 3.2 --- Routine cell line maintenance --- p.35 / Chapter 3.3 --- Drug treatments --- p.35 / Chapter 3.4 --- Total RNA extraction --- p.35 / Chapter 3.5 --- Genomic DNA extraction --- p.36 / Chapter 3.6 --- General techniques --- p.37 / Chapter 3.6.1 --- Gel electrophoresis --- p.37 / Chapter 3.6.2 --- DNA and RNA quantification --- p.37 / Chapter 3.6.3 --- LB medium and LB plate preparation --- p.38 / Chapter 3.6.4 --- Plasmid extraction --- p.38 / Chapter 3.6.4a --- Mini-scale preparation of plasmid DNA --- p.38 / Chapter 3.6.4b --- Large-scale preparation of endotoxin-free plasmid DNA --- p.39 / Chapter 3.6.5 --- DNA sequencing --- p.39 / Chapter 3.7 --- Reverse transcription-PCR (RT-PCR) --- p.40 / Chapter 3.7.1 --- Reverse transcription (RT) --- p.40 / Chapter 3.7.2 --- Semi-quantitative RT-PCR --- p.41 / Chapter 3.8 --- Methylation analysis --- p.42 / Chapter 3.8.1 --- Sodium bisulfite modification of DNA --- p.42 / Chapter 3.8.2 --- CpG island analysis --- p.42 / Chapter 3.8.3 --- Methylation-specific PCR (MSP) --- p.43 / Chapter 3.8.4 --- Bisulfite genomic sequencing (BGS) --- p.44 / Chapter 3.9 --- Construction of expression plasmids --- p.45 / Chapter 3.9.1 --- Construction of the MGC80-expressing vector --- p.45 / Chapter 3.9.2 --- Construction of the TUSC14-expressing vector --- p.46 / Chapter 3.10 --- Functional analyses --- p.47 / Chapter 3.10.1 --- Monolayer colony formation assay --- p.47 / Chapter 3.10.2 --- Soft agar assay --- p.48 / Chapter 3.11 --- Statistical analysis --- p.49 / Chapter Chapter 4 --- Results --- p.50 / Chapter 4.1 --- Identification of 5qll-ql2 and 14q32.2-q32.32 as frequently deleted regions in NPC by aCGH --- p.50 / Chapter 4.2 --- Identification of novel candidate TSGs at chromosome 5qll-ql2 through integrative genomics and epigenetics --- p.51 / Chapter 4.2.1 --- Expression profiling of the candidate genes at 5ql l-ql2 in NPC cell lines --- p.51 / Chapter 4.2.2 --- MGC80 as a target of study at 5ql2 --- p.54 / Chapter 4.2.2a --- Ubiquitous expression in normal human tissues and frequent down-regulation of MGC80 in multiple tumor cell lines --- p.54 / Chapter 4.2.2b --- Methylation analysis of MGC80 --- p.56 / Chapter 4.2.2c --- Restoration of MGC80 expression after pharmacologic and genetic demethylation --- p.59 / Chapter 4.2.2d --- Functional study of MGC80 in multiple carcinomas --- p.61 / Chapter 4.2.2e --- Discussion --- p.63 / Chapter 4.2.3 --- TUSC14 as a target of study at 5ql2 --- p.67 / Chapter 4.2.3a --- TUSC14 was broadly expressed in normal human tissues and frequently down-regulated in multiple tumor cell lines --- p.67 / Chapter 4.2.3b --- Methylation analysis of TUSCI4 --- p.69 / Chapter 4.2.3c --- Pharmacologic and genetic demethylation reactivated TUSC14 expression --- p.72 / Chapter 4.2.3d --- Functional study ofTUSC14 in multiple carcinomas --- p.74 / Chapter 4.2.3e --- Discussion --- p.76 / Chapter 4.3 --- Identification of candidate TSGs at chromosome 14q32 through integrative genomics and epigenetics --- p.80 / Chapter 4.3.1 --- Expression profiling of the candidate genes at 14q32 in NPC cell lines --- p.80 / Chapter 4.3.2 --- DLK1 as a target of study at 14q32 --- p.82 / Chapter 4.3.2a --- Expression analysis of DLK1 in normal tissues and NPC cell lines --- p.82 / Chapter 4.3.2b --- Methylation analysis ofDLKl in NPC --- p.83 / Chapter 4.3.2c --- Restoration of DLK1 expression after pharmacologic demethylation --- p.84 / Chapter 4.3.2d --- Functional study ofDLKl in NPC --- p.85 / Chapter 4.3.2e --- Discussion --- p.87 / Chapter Chapter 5 --- General discussion --- p.92 / Chapter Chapter 6 --- Summary --- p.99 / Chapter Chapter 7 --- Future study --- p.101 / Reference list --- p.103

EDD (E3 isolated by differential display), located at chromosome 8q22.3, is the human homologue of the Drosophila melanogaster tumour suppressor gene 'hyperplastic discs'. Edd null mice and hyd mutants display embryonic lethality. EDD is also a multifunctional HECT family E3 ubiquitin protein-ligase, with reported roles in both progesterone action and the DNA damage response. To investigate the possible involvement of EDD in human cancer, several cancer types were analysed for allelic gain or loss (allelic imbalance, AI) at the EDD locus. AI of the EDD locus was most frequent in the serous subtype of ovarian cancer (16/22, 73%) and common in other cancers, including breast cancer (31%). AI is likely to represent amplification of the EDD gene locus rather than loss of heterozygosity, as quantitative RT-PCR and immunohistochemistry showed that EDD mRNA and protein are frequently overexpressed in breast and ovarian cancers. These data imply a potential role for EDD in cancer progression. However, depletion of EDD from cells in culture by RNA interference had very little effect on proliferation and cell survival. To identify EDD-regulated pathways, transcript analysis was performed on EDD-depleted cells. The results suggested that EDD modulates cell-cell communication and the actin cytoskeleton. Consistent with transcript analysis, depletion of EDD from two normal breast cell lines (HMEC-184 and MCF-10A) resulted in altered cell morphology, with decreased cell-cell contacts. This was concurrent with altered beta-catenin (an integral component of adherens junctions) at cell-cell contacts, which was also observed in the developing blood vessels of Edd null mice. Interestingly, total cellular beta-catenin levels were not affected. Furthermore, EDD depletion resulted in a decrease in expression of the cytoskeletal regulators twinfilin and R-RAS, with a simultaneous decrease in MAPK (ERK1 and ERK2) activity. Consistent with disruption of adherens junctions, EDD-depleted mammary acini lost tissue coordination and polarity. These data provide a significant advance in our knowledge of EDD, both in its role in regulating the organisation of cells into higher structures and its potential role in the development of cancer. This has relevance to an understanding of embryonic development and the role of tissue homeostasis in cancer progression.

IKK[alpha] is a 85KD serine/threonine protein kinase and a subunit of the IKK complex, which contains IKK[alpha], IKK[beta], and IKK[gamma]. IKK[alpha] and IKK[beta] are highly conserved and they contain three functional domains of kinase domain, leucine zipper (LZ), and helix-loop-helix (HLH). Although IKK[alpha] and IKK[beta] can phosphorylate IκB proteins in vitro, IKK[alpha] and IKK[beta] have distinct physiological functions during mouse development. Genetic studies showed that IKK[alpha] is essential for embryonic skin development in mice. Mice deficient in IKK[alpha] display a hyperplastic epidermis that lacks terminal differentiation, resulting a death soon after birth because of the severely impaired skin. Recently, we reported a reduction in IKK[alpha] expression and identified somatic Ikk[alpha] mutations in a high proportion of poorly differentiated human squamous cell carcinomas (SCCs) (Liu et al., 2006). The aim of this study is to investigate the novel role of IKK[alpha] in skin carcinogenesis. We firstly examined IKK[alpha] expression and Ikk[alpha] mutations in human SCCs and found a reduction of IKK[alpha] in poorly differentiated human SCCs and identified somatic Ikk[alpha] mutations in exon 15 of Ikk[alpha] in human SCCs. We then examined the susceptibility of Ikk[alpha] hemizygotes to chemical carcinogeninduced skin carcinogenesis. In this chemical carcinogen-induced skin carcinogenesis setting, 7,12-dimethylbenz[a]anthracene (DMBA) induces Ras mutations and 12-Otetradecanoyl-phorbol-13-acetate (TPA) promotes Ras-initiated cell proliferation. We found two times more papillomas and eleven times more carcinomas in Ikk[alpha superscript +/-] mice than in Ikk [alpha] superscript +/+] mice induced by DMBA/TPA. Ikk[alpha superscript +/-] mice developed larger and earlier tumors than did Ikk[alpha superscript +/+] mice. Poorly differentiated carcinomas expressed low levels of IKK[alpha]. Ninety five percent of the Ikk[alpha superscript +/-] carcinomas and 44% of the Ikk[alpha superscript +/-] papillomas lost the remaining wild type Ikk[alpha] allele. This result indicates that the remaining one wild type Ikk[alpha] allele is important for preventing malignant carcinoma conversion. Also Ikk[alpha] mutations were detected in these skin tumors. Reduced IKK[alpha] was found to enhance TPA-induced mitogenic and angiogenic activities in mouse skin. Taken together, these results suggest that reduction of IKK[alpha] expression provides a selective growth advantage, which cooperates with DMBA-initiated Ras activity to promote skin carcinogenesis. In addition, we observed a small group of FVB female Ikk [alpha superscript +/-] mice for 1.5 years and found that 12/ 24 mice developed various spontaneous tumors including mammary gland carcinomas, uterine and ovary tumors, and dermal fibrosacomas. Somatic Ikk[alpha] mutations, elevated IKK/ NF[subscript -k]B and extracellular signal-regulated kinases (ERK) activities and elevated cyclin D1 levels were detected in these spontaneous tumors. These results suggest that these molecular alterations may contribute to the development of these tumors although the precise role of the down-regulation of IKK in the development of the tumors remains to be determined. Overall, our data and other published results suggest that IKK[alpha] is a new tumor suppressor in men and mice. / text

Precancerous lesions from a variety of human tissues display markers of DNA damage suggesting that genetic instability occurs early during the process of carcinogenesis. Consistent with this, several oncogenes can activate ATM and other components of the DNA damage response pathway when expressed in cultured cells. Here we demonstrate that preneoplastic epithelial tissues from four different transgenic mouse models expressing the oncogenes c-myc, SV40 T antigen, human papilloma virus (HPV) E7, or E2F3a display [gamma]-H2AX foci and other markers of DNA damage. Moreover, transgenic expression of these oncogenes leads to increased levels of damaged DNA as measured by the comet assay. In at least the Myc transgenic model, the formation of [gamma]-H2AX foci is dependent on functional ATM. Inactivation of Atm also impairs p53 activation and reduces the level of apoptosis observed in transgenic tissue overexpressing Myc. This correlates with accelerated tumor development in Myc transgenic mice lacking ATM. To understand the mechanism by which oncogenes induce DNA damage, we employed an adenoviral overexpression system. Under conditions in which Myc or E2F3a induced replication is inhibited, we see a reduction in the DNA damage induced by these oncogenes both by comet assay and levels of [gamma]-H2AX. Moreover, Myc and E2F3a induced increased levels of the Cdt1 protein, a replication origin- licensing factor implicated in aberrant DNA replication. Taken together, these findings suggest that deregulated oncogenes induce unscheduled DNA replication leading to DNA damage and activation of the ATM DNA damage response pathway, which is important for the activation of p53, induction of apoptosis and the suppression of tumorigenesis. / text

published_or_final_version / Medical Sciences / Master / Master of Medical Sciences