Wang, Shiyan. / Thesis (Ph.D.)--Chinese University of Hong Kong, 2011. / Includes bibliographical references (leaves 123-139). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Abstract also in Chinese.

Genes identified by SAGE may serve as potential prognostic marker or therapeutic target. 14-3-3 sigma is a putative tumor suppressor and can be induced in response to DNA damage following irradiation, leading to cell cycle arrest in G2/M in human cancer cells. Our SAGE results revealed that 14-3-3 sigma expression is significantly downregulated in C666-1 cells. The study of 72 primary NPCs showed that an increased expression of 14-3-3 sigma was associated with a poorer clinical outcome in terms of shorter overall survival (OS; p=0.0297) and shorter disease free survival (DFS; p=0.042) using univariate analysis. Hence, 14-3-3 sigma may be used as an independent prognostic marker for NPC patients. / In conclusion, a NPC transcription profile has been successfully generated and several candidate NPC-associated genes have been identified by Serial Analysis of Gene Expression (SAGE) and NPC transcriptome map. These novel findings lead to better understanding of the molecular basis of NPC development and provide a foundation for discovery of new therapeutic strategies. / Nasopharyngeal carcinoma (NPC) is one of the most prevalent cancers among Southern Chinese. To better understand the genetic basis of this disease, Serial Analysis of Gene Expression (SAGE) was performed to investigate the transcriptional profiles of an EBV-positive NPC cell line (C666-1) and a normal NP outgrowth (NP4). A total of 102,059 SAGE tags were extracted in both libraries and 250 genes with 10-fold or more differential expression were found in NPC cells compared to normal NP cells. Eleven differentially expressed genes identified by SAGE were selected for confirmation using real time RT-PCR. The transcripts for 5 of the 11 genes, CD 74, Transcriptional intermediary factor 1, Ferritin 1, Claudin 4, and fatty acid synthase were overexpressed in NPC cells. Conversely, the remaining transcripts including Keratin 17, Keratin 5, S100 calcium-binding A2, Cystatin A, 14-3-3 sigma and Caveolin 1 were underexpressed in NPC cells. The aberrant expression of CD74, Claudin 4, Fatty acid synthase, 14-3-3 sigma, Caveolin 1 were further validated by immunohistochemistry on 20 NPC patients. / On the other hand, fatty acid synthase (FASN), a key enzyme for de novo lipogenesis, is a putative therapeutic target in treating NPC. Immunohistochemical studies showed upregulation of FASN in 20.8% (15/72) of the NPC cases compared with the adjacent normal NP epithelium. In addition, FASN expression also had prognostic significance in predicting the outcome of patients after radiotherapy, as high levels of FASN expression were associated with worse overall survival (OS, p=0.032) and disease free survival (DFS, p=0.002) in NPC patients. FASN inhibitors, such as C75 which inhibit cell growth via cell cycle arrest in G2/M phase, are potential chemotherapeutic agents in treating NPC. / The genome-wide quantitative analysis of gene expression by SAGE with matched chromosomal positions enables the construction of a transcriptome map of NPC. A total of 8 and 29 overexpressed and underexpressed gene clusters were observed, respectively. Some novel regions that have never been illustrated in previous reports such as amplification regions at 2p11.2-p25.1, 2q33-q37, 9q22-q34, 17p11.2-p13.2 and deletion regions at 1p12-p31.2, 1q25-q42.12, 2q21.3-q33, 8p21.1-p22, 9q33-q34.3, 10q23.3-q26.3, 12p13, 16p13, 17q23.2-q25, 19p13, 19q12-q13.2, 20p11-p13, 22q13, Xp11.2-p11.4, and Xq26-q28 were also identified. A candidate tumor suppressor gene named MEG3 has been found within an underexpressed region at 14q32.2 in the NPC transcriptome map. Our FISH analysis revealed that chromosome loss at 14q32 is associated with hypermethylation of MEG3 promoter region in 9/13 (75%) of NPC patients. Loss of imprinting is the major mechanism that governs the MEG3 expression. Moreover, transient transfection of one of the MEG3 isoforms (accession no. AF119863) could obviously inhibit cell colony formation of NPC cells. Taken together, MEG3 gene on chromosome 14q32.2 might act as a tumor suppressor in NPC. / Chan, Yat Yee. / "March 2008." / Adviser: Lo Kwok Wai. / Source: Dissertation Abstracts International, Volume: 70-03, Section: B, page: 1605. / Thesis (Ph.D.)--Chinese University of Hong Kong, 2008. / Includes bibliographical references (p. 196-225). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Electronic reproduction. [Ann Arbor, MI] : ProQuest Information and Learning, [200-] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Abstracts in English and Chinese. / School code: 1307.

Zhang, Bin. / Thesis (Ph.D.)--Chinese University of Hong Kong, 2013. / Includes bibliographical references (leaves 154-187). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Abstract also in Chinese.

Xu, Jiaying. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2013. / Includes bibliographical references (leaves 98-107). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Abstracts also in Chinese.

In this study, we investigated the mechanisms by which PBRM1 functions as a tumor suppressor in clear cell renal cell carcinoma. PBRM1, also known as BAF180 or Polybromo, is a member of the PBAF SWI/SNF chromatin remodeling complex. Cancer sequencing studies have revealed that SWI/SNF components are widely mutated in cancer. PBRM1 is recurrently mutated in various human malignancies, but it has a particularly high mutation rate in clear cell renal cell carcinoma: ~40% of clear cell renal cell carcinomas have a PBRM1 mutation, making it the second most highly mutated gene in clear cell renal cell carcinoma behind VHL. Although many recent studies have looked at how other SWI/SNF components function in cancer control, relatively little is known about the tumor suppressive mechanisms of PBRM1 in clear cell renal cell carcinoma. To investigate PBRM1 function, we manipulated its expression in clear cell renal cell carcinoma cell lines. In cell lines with intact PBRM1, we stably knocked down its expression using shRNA. In a cell line with mutant PBRM1, we stably restored expression of the wild-type protein. We found that PBRM1 deficiency significantly enhanced the growth properties of cells, but only when the cells were grown under stressful conditions, such as reduced serum or a 3-D culture environment. To investigate genes and pathways influenced by PBRM1 that may confer this growth advantage, we compared gene expression differences in the clear cell renal cell carcinoma cell lines and murine embryonic fibroblasts with or without PBRM1. We found that PBRM1 regulated numerous cancer-related genes and pathways. One gene, ALDH1A1, was consistently upregulated with PBRM1 deficiency across our cell lines. Further expression analysis using two different clear cell renal cell carcinoma primary tumor datasets revealed that PBRM1 mutation in primary tumors was also associated with higher ALDH1A1 levels. ALDH1A1, or aldehyde dehydrogenase 1, is part of the retinoic acid metabolic pathway and irreversibly converts retinaldehyde to retinoic acid. It functions in hematopoietic stem cell development, white versus brown fat programming, and insulin signaling. Numerous studies have also identified ALDH1A1 as a marker of tumor-initiating cells, also known as cancer stem cells. Not much is known about the regulation of ALDH1A1 expression in cancer, and it has not previously been linked to PBRM1 or SWI/SNF. We confirmed that stable knockdown of PBRM1 in clear cell renal cell carcinoma cell lines resulted in higher ALDH1A1 mRNA and protein expression, and also higher ALDH1-class enzyme activity. Alternatively, re-expression of wild-type PBRM1, but not cancer-associated mutant PBRM1, lowered ALDH1A1 expression and activity in the PBRM1-mutant line. Additionally, inhibiting ALDH1A1 or knocking it down in the context of PBRM1 deficiency reduced anchorage-independent growth, while over-expressing ALDH1A1 in the PBRM1-normal setting increased tumorsphere-forming capacity. These results suggest that ALDH1A1 is not only a marker of tumor-initiating cells, but can also increase the tumorigenic potential of cells. Based on our gene expression analysis, we additionally explored PBRM1 regulation of the EGFR and IFN pathways. PBRM1 decreased total EGFR protein levels and dampened downstream signaling. These changes had functional consequences, as PBRM1 deficiency led to faster growth in response to EGF stimulation. However, it did not create a setting of oncogenic addiction, as PBRM1 deficient cells were also more resistant to EGFR inhibition. Alternatively, PBRM1 deficiency reduced basal and IFNα-induced levels of IFI27, a pro-apoptotic interferon response gene, and made cells more resistant to growth inhibition by IFNα. PBRM1 mutations in cancer would thus be expected to have wide-ranging effects on a cell, and the targeting of any one specific downstream pathway might have limited efficacy. Finally, we investigated the molecular mechanisms of how PBRM1 deficiency could alter transcription, keeping in mind that PBRM1 is one subunit of the larger PBAF complex. In our clear cell renal cell carcinoma cell lines, we found that mRNA and protein levels of another PBAF-specific subunit, ARID2, increased with PBRM1 deficiency. PBRM1 mutation in primary tumors was also associated with significantly higher ARID2 expression. Immunoprecipitation and glycerol gradient fractionation experiments suggested that more ARID2 may associate with the SWI/SNF components BRG1 and SNF5 after PBRM1 knockdown. ARID2 ChIP-seq analysis revealed that this remnant PBAF-like complex was bound to fewer locations in the genome, and its binding locations were broadly redistributed. Both gained and lost ARID2 binding were associated with differential gene expression, of both upregulated and downregulated genes, indicating that the genomic context influences whether PBAF-binding is activating or repressive. Interestingly, we also found that ARID2 was required for some of the pro-tumorigenic changes associated with PBRM1 deficiency, such as upregulation of ALDH1A1 and EGFR levels, but not others, such as decreased IFI27 levels, implying alternative modes of transcriptional regulation. In total, this study implicates PBRM1 in the regulation of numerous cancer-related genes and pathways in clear cell renal cell carcinoma. PBRM1 mutation would alter the genomic binding of a residual PBAF-like complex containing ARID2, leading to transcriptional changes that promote tumor formation and growth. A better understanding of this oncogenic mechanism may reveal novel therapeutic opportunities.

An important hallmark of cancer is the inactivation of tumor suppressor genes. The most common genetic alteration in cancer is the mutation of the TP53 gene occurring in about half of all cancers, but very little progress has been made on how to therapeutically target the signaling defects in these cancers. Additionally, the PTEN tumor suppressor is mutated in a wide variety of cancer types, and its expression is often lost in the absence of mutation. PTEN is a haploinsufficient tumor suppressor that exhibits dose-dependent effects in cells. In the context where PTEN is lost or downregulated, PI3K signaling and downstream signaling through AKT is overactive, leading to an increase in cell growth and proliferation, among other effects. Acting as both a protein and lipid phosphatase, loss of PTEN also affects the PI3K-independent signaling of PTEN, and results in an increase of migration and invasion phenotypes. Importantly, PTEN transcript level is the key determinant for PTEN protein expression, and downregulation of PTEN is part of a poor-prognosis gene expression signature in breast cancer. Downregulation of tumor suppressor gene expression represents a reversible change that is often sufficient to drive tumorigenesis. However, our understanding of the broad molecular mechanisms by which the expression of these tumor suppressors is lost remains limited, but is required to develop effective therapeutic strategies to target malignancies driven by tumor suppressor loss. In Chapter 2, we characterize the problem of transcriptional downregulation of PTEN in breast cancer. We investigate the expression of PTEN in various normal and tumor cells at both the transcript and protein level. We identify various model systems that we believe are suitable to model normal PTEN expression and the PTEN downregulation that mimics what is observed in tumors. We employ a sophisticated approach that couples RNA-sequencing with Nanostring nCounter analysis in order to obtain a detailed and thorough transcriptional profile of the PTEN and pseudogene PTENP1 genomic loci, as well as expression of the poor-prognosis gene signature associated with PTEN downregulation. In this study, we obtained an understanding of the changes in the PTEN transcriptional profile that occur in the progression from normal to cancer, and we believe this approach could be applied to other key tumor suppressor genes. In Chapter 3, we discovered that basally expressed p53 maintains expression of thirteen well-validated tumor suppressors. p53 is expressed at low levels under normal, low-stress conditions, and is expressed at much higher levels under enhanced stress, leading to the activation of stress-response genes. We begin the study by highlighting an association between TP53 mutation and downregulation of PTEN expression. Upon performing chromatin immunoprecipitation coupled with next generation sequencing for p53 under normal, low-stress conditions, we found that p53 binds in the vicinity of thirteen tumor suppressor genes, including PTEN. Basally expressed p53 binds to classic consensus binding sites in enhancers and promoters of target tumor suppressors to maintain their expression at baseline. CRISPR/Cas9-mediated knockout of the endogenous basal p53 binding site upstream of PTEN led to a decrease in PTEN expression and an increase in tumorigenic phenotypes. Given that mutation of TP53 leads to tumorigenesis in mice, but loss of p53 stress-response targets or loss of the ability of p53 to activate these stress-response targets does not lead to spontaneous tumorigenesis, it is likely that these tumor suppressor targets of basal p53 contribute to p53-mediated tumor suppression. In Chapter 4, we identified yet another mechanism by which transcriptional repression of PTEN occurs in triple-negative breast cancer (TNBC) through polycomb repressive complex 2 (PRC2)-mediated repression of the PTEN promoter and upstream regulatory region. Previous research has shown that mutated NOTCH1 represses PTEN through the HES-1 transcription factor in acute myeloid leukemia (AML), and that NOTCH translocations are frequent in TNBC and are sufficient for transformation in vitro. We discovered that NOTCH1 and NOTCH2 mutations and translocations correlate with PTEN downregulation by immunohistochemistry in a cohort of TNBC cases. The TNBC cell line exhibiting PRC2-mediated repression of PTEN also harbors a SEC22B-NOTCH2 translocation that creates a gene product resembling the NOTCH2 intracellular domain. The NOTCH target HES-1 co-localizes on the PTEN promoter with EZH2 (the lysine methyltransferase involved in PRC2-mediated transcriptional repression), and knockdown of NOTCH2 in this cell line led to decreased expression of EZH2, and restoration of PTEN expression at the transcript and protein level. We also demonstrated that EZH2 inhibitors, HDAC inhibitors, and DNA hypomethylating agents robustly restore PTEN transcript levels. Taken together, these results elucidate another mechanism by which PTEN is transcriptionally repressed in the highly aggressive and poor-prognosis TNBC subtype of breast cancer that may be applicable to other cancer types. The results also suggest that this repression is reversible by pharmacological approaches, highlighting a promising therapeutic avenue. Taken together, the studies presented in this thesis begin to unravel the complex mechanisms of transcriptional repression of tumor suppressor genes in cancer. As is the case with PTEN and p53, multiple regulatory mechanisms can influence expression in combination or in a context-dependent manner. The loss of expression of tumor suppressor genes is one of the key hallmarks of cancer, yet very few of the therapeutic approaches used in the clinic today aim to restore tumor suppressor expression. Our results demonstrate proof of concept that restoration of tumor suppressor expression is a plausible and promising therapeutic approach for many different types of cancer, but requires a detailed understanding of the underlying molecular mechanisms of transcriptional regulation.

Mak, Ko Fung. / Thesis submitted in: July 2003. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2004. / Includes bibliographical references (leaves 113-138). / Abstracts in English and Chinese. / Chapter CHAPTER I: --- INTRODUCTION --- p.1 / Chapter I. --- AIM OF STUDY --- p.1 / Chapter II. --- LITERATURE REVIEW --- p.2 / Chapter 1. --- Epidemiology --- p.2 / Chapter 2. --- Etiology --- p.4 / Chapter A. --- Heredity --- p.4 / Chapter i. --- Family History --- p.4 / Chapter ii. --- Inherited Predisposition --- p.4 / Chapter B. --- Hormonal --- p.7 / Chapter C. --- Environmental --- p.9 / Chapter i. --- Diet --- p.9 / Chapter ii. --- Radiation --- p.10 / Chapter iii. --- Physical Activity --- p.11 / Chapter 3. --- Histopathology --- p.12 / Chapter 4. --- Clonality Nature of Cancer --- p.13 / Chapter 5. --- "Knudson ""two-hit"" Hypothesis on Cancer Development" --- p.14 / Chapter 6. --- Molecular Genetic Studies of Breast Cancer --- p.15 / Chapter A. --- Loss of Heterozygosity --- p.16 / Chapter B. --- Comparative Genomic Hybridization --- p.19 / Chapter C. --- Epigenetic Changes --- p.20 / Chapter 7. --- Genetic Changes in Breast Cancer --- p.21 / Chapter A. --- Chromosome 1 --- p.21 / 14-3-3σ Gene --- p.21 / Chapter B. --- Chromosome 3 --- p.22 / Chapter i. --- Retionoic Acid Receptor p2 Gene --- p.22 / Chapter ii. --- Fragile Histidine Traid Gene --- p.24 / Chapter iii. --- Ras Associated Domain Family 1A Gene --- p.25 / Chapter iv. --- Thyroid Hormone Receptor β1 Gene --- p.26 / Chapter C. --- Chromosome 5 --- p.27 / Adenomatous Polyposis Coli Gene --- p.27 / Chapter D. --- Chromosome 6 --- p.28 / Estrogen Receptor Gene --- p.28 / Chapter E. --- Chromosome 9 --- p.29 / p16 Gene --- p.29 / Chapter F. --- Chromosome 13 --- p.30 / Chapter i. --- BRCA2 Gene --- p.31 / Chapter ii. --- Retinoblastoma Gene --- p.32 / Chapter G. --- Chromosome 16 --- p.33 / E-cadherin Gene --- p.33 / Chapter H. --- Chromosome 17 --- p.34 / Chapter i. --- TP53 Gene --- p.34 / Chapter ii. --- BRCA1 Gene --- p.36 / Chapter CHAPTER II: --- MATERIALS AND METHODS --- p.38 / Chapter I. --- PATIENTS AND SPECIMENS --- p.38 / Chapter II. --- FROZEN SECTIONS AND MICRODISSECTION --- p.41 / Chapter III. --- DNA EXTRACTION --- p.43 / Chapter IV. --- ALLELOTYPING --- p.44 / Chapter 1. --- Polymerase Chain Reaction --- p.44 / Chapter 2. --- Electrophoresis --- p.45 / Chapter 3. --- Data Analysis --- p.46 / Chapter CHAPTER III: --- RESULTS --- p.49 / Chapter I. --- ALLELOTYPING ANALYSIS --- p.49 / Chapter II. --- FREQUENCY OF LOH --- p.68 / Chapter III. --- FRACTIONAL ALLELIC LOSS --- p.70 / Chapter IV. --- MINIMAL DELETION REGIONS --- p.72 / Chapter 1. --- Chromosome 6q --- p.83 / Chapter 2. --- Chromosome 8p --- p.83 / Chapter 3. --- Chromosome 8q --- p.84 / Chapter 4. --- Chromosome 10q --- p.84 / Chapter 5. --- Chromosome 13q --- p.85 / Chapter 6. --- Chromosome 14q --- p.85 / Chapter 7. --- Chromosome 16q --- p.86 / Chapter V. --- MICROSATELLITE INSTABILITY --- p.86 / Chapter CHATPER IV: --- DISCUSSION --- p.88 / Chapter I. --- COMPARISONS OF CHROMOSOMAL ALTERATIONS --- p.88 / Chapter II. --- MICROSATELLITE INSTABILITY --- p.92 / Chapter III. --- CHROMOSOMAL GAINS AND LOSSES --- p.93 / Chapter IV. --- CHROMOSOME 17 --- p.95 / Chapter V. --- MINIMAL DELETION REGIONS --- p.96 / Chapter 1. --- Chromosome 6q --- p.97 / Chapter 2. --- Chromosome 8p --- p.99 / Chapter A. --- 8p23.3.-22 --- p.99 / Chapter B. --- 8p21.3-12 --- p.101 / Chapter C. --- 8p22-21 --- p.103 / Chapter 3. --- Chromosome 8q --- p.104 / Chapter 4. --- Chromosome 10q --- p.106 / Haploinsufficiency and PTEN --- p.107 / Chapter 5. --- Chromosome 13q --- p.108 / Chapter 6. --- Chromosome 14q --- p.109 / Chapter 7. --- Chromosome 16q --- p.110 / Chapter CHAPTER V: --- CONCLUSION --- p.112 / REFERENCES --- p.113

Ng Yeung Lam. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2005. / Includes bibliographical references (leaves 180-209). / Abstracts in English and Chinese. / acknowledgements --- p.i / abstract --- p.ii / abstract in chinese --- p.vi / table of contents --- p.ix / list of tables --- p.xiii / list of figures --- p.xi v / list of abbreviations --- p.xvi / Chapter 1 --- chapter1 introduction and literature review --- p.1 / Chapter 1.1 --- Introduction of brain tumors --- p.1 / Chapter 1.2 --- Oligodendroglial tumors (OTs) --- p.3 / Chapter 1.2.1 --- Oligodendroglioma (OD) and anaplastic oligodendroglioma (AOD) --- p.3 / Chapter 1.2.1.1 --- WHO's definition and grading --- p.3 / Chapter 1.2.1.2 --- "Incidence, age, sex distribution, tumor location and survival rate" --- p.3 / Chapter 1.2.1.3 --- Clinical presentation --- p.4 / Chapter 1.2.1.4 --- Macroscopy and histopathology --- p.4 / Chapter 1.2.1.5 --- Immunohistochemistry --- p.5 / Chapter 1.2.1.6 --- Treatment --- p.6 / Chapter 1.2.2 --- Oligoastrocytoma (OA) and anaplastic oligoastrocytoma (AOA) --- p.11 / Chapter 1.2.2.1 --- WHO's definition and grading --- p.11 / Chapter 1.2.2.2 --- "Incidence, age, sex distribution, tumor location and survival rate" --- p.12 / Chapter 1.2.2.3 --- Clinical features --- p.12 / Chapter 1.2.2.4 --- Macroscopy and histopathology --- p.12 / Chapter 1.3 --- Overview of Genetic and Epigenetic Aberrations of OTs --- p.14 / Chapter 1.3.1 --- Chromosomal and genetic aberrations in OTs --- p.14 / Chapter 1.3.2 --- Candidate regions and genes on 1 p --- p.15 / Chapter 1.3.3 --- Candidate regions and genes on 19q --- p.20 / Chapter 1.3.4 --- Other aberrations in WHO grade II OTs --- p.24 / Chapter 1.3.5 --- Progression-associated aberrations in ODs --- p.25 / Chapter 1.3.6 --- Chromosomal and genetic aberrations in OAs --- p.29 / Chapter 1.4 --- Correlation of genetic alterations with response to therapy and survival --- p.31 / Chapter 1.4.1 --- Response to PCV chemotherapy correlates with lp and combined lp/19q status in patients with AODs --- p.31 / Chapter 1.4.2 --- Survival of patients with AODs correlates with lp/19q status --- p.32 / Chapter 1.4.3 --- WHO grade II ODs behavior and lp/19q status --- p.32 / Chapter 1.4.4 --- Response to other therapies (temozolomide and radiotherapy) and lp/19q status in patients with ODs --- p.33 / Chapter 1.4.5 --- lp and 19q loss in OAs and diffuse astrocytomas --- p.34 / Chapter 1.5 --- Microarray-based expression profiling of OTs --- p.35 / Chapter 1.6 --- Description of p73 protein --- p.37 / Chapter 1.6.1 --- Introduction of p73 --- p.37 / Chapter 1.6.2 --- p73: gene structure and splicing variants --- p.37 / Chapter 1.6.3 --- Signaling in p73 --- p.40 / Chapter 1.6.4 --- Regulation ofp73 protein stability and transcriptional activity --- p.43 / Chapter 1.6.4.1 --- Regulation by DNA damage --- p.43 / Chapter 1.6.4.2 --- Regulation by oncogenes --- p.44 / Chapter 1.6.4.3 --- Interaction with viral proteins --- p.44 / Chapter 1.6.5 --- Role of p73 in the nervous system --- p.45 / Chapter 1.6.6 --- p73 in cancer --- p.45 / Chapter 1.6.6.1 --- p73 knockout mice --- p.45 / Chapter 1.6.6.2 --- Alteration of p73 expression in human cancers --- p.46 / Chapter 1.6.7 --- p73 and chemosensitivity --- p.50 / Chapter CHAPTER2 --- AIMS OF STUDY --- p.51 / Chapter CHAPTER3 --- MATERIALS AND METHODS --- p.53 / Chapter 3.1 --- Tumor and blood samples --- p.53 / Chapter 3.2 --- Cell culture --- p.53 / Chapter 3.3 --- DNA extraction from frozen tissues and blood samples --- p.54 / Chapter 3.4 --- Detection of allelic loss of chromosome lp --- p.58 / Chapter 3.4.1 --- LOH analysis --- p.58 / Chapter 3.4.2 --- Fluorescence in situ Hybridization (FISH) analysis on Paraffin and Frozen Sections --- p.60 / Chapter 3.6 --- DNA sequencing analysis --- p.62 / Chapter 3.7 --- Analysis of Methylation --- p.63 / Chapter 3.7.1 --- Bisulfite sequencing --- p.63 / Chapter 3.7.2 --- Methylation-specific polymerase chain reaction (MSP) --- p.66 / Chapter 3.8 --- Northern Blot analysis --- p.68 / Chapter 3.9 --- RNA isolation and cDNA preparation --- p.70 / Chapter 3.10 --- Laser microdissection and RNA extraction from microdissected tumor cells --- p.71 / Chapter 3.10.1 --- Conventional RT-PCR --- p.71 / Chapter 3.11 --- Primer design for TP73 and its isoforms --- p.74 / Chapter 3.12 --- Real-time RT-PCR --- p.77 / Chapter 3.12.1 --- Real-time RT-PCR for TP73 and its isoforms --- p.78 / Chapter 3.12.2 --- Real-time RT-PCR for KIAA0495 --- p.79 / Chapter 3.13 --- Statistical analyses --- p.81 / Chapter CHAPTER4 --- RESULTS --- p.82 / Chapter 4.1 --- Genes annotated in the minimally deleted regions --- p.82 / Chapter 4.2 --- Expression analyses of TP73 and its isoforms in ODs by quantitative real-time RT-PCR --- p.85 / Chapter 4.3 --- Methylation analysis of TP73 in ODs by methylation sensitive PCR (MSP) --- p.97 / Chapter 4.4 --- A rapid screen of candidate genes for aberrant expression in microdissected tumors --- p.100 / Chapter 4.5 --- Quantitative real-time RT-PCR of KIAA0495 gene --- p.103 / Chapter 4.6 --- Mutation analysis of KIAA0495 gene --- p.110 / Chapter 4.7 --- Methylation analysis of KIAA0495 in ODs by bisulfite sequencing…… --- p.112 / Chapter 4.8 --- Detection of allelic loss of lp by LOH analysis and interphase FISH --- p.121 / Chapter 4.9 --- Two-hit inactivation of KIAA0495 gene in ODs --- p.126 / Chapter 4.10 --- Tissue distribution of KIAA0495 gene --- p.130 / Chapter 4.11 --- Bioinformatics of KIAA0495 --- p.133 / Chapter CHAPTER5 --- DISCUSSION --- p.146 / Chapter 5.1 --- Expression analysis of TP73 and its isoforms in ODs by isoform-specific RT-PCR --- p.148 / Chapter 5.2 --- Methylation status ofTP73 in ODs --- p.153 / Chapter 5.3 --- A rapid screening of candidate genes for aberrant expressionin microdissected tumors --- p.156 / Chapter 5.4 --- Expression pattern of KIAA0495 mRNA in a large cohort of ODs --- p.157 / Chapter 5.5 --- No somatic mutation in coding region of KIAA0495 --- p.158 / Chapter 5.6 --- Methylation status of putative promoter region of KIAA0495 in ODs --- p.159 / Chapter 5.7 --- Status of chromosome lp in ODs --- p.161 / Chapter 5.8 --- Two-hit inactivation of KIAA0495 gene in ODs by promoter hypermethylation and allelic loss of lp --- p.162 / Chapter 5.9 --- Evaluation of expression of KIAA0495 gene as a marker for the response to chemotherapy and prognostic marker in patients with OTs --- p.164 / Chapter 5.10 --- Tissue distribution of KIAA0495 --- p.166 / Chapter 5.11 --- "KIAA0495 cDNA sequence, protein sequence and potential functional features" --- p.167 / Chapter 5.12 --- Candidate tumor suppressor genes on lp in other type of tumors with loss of lp --- p.171 / Chapter CHAPTER6 --- CONCLUSIONS --- p.174 / Chapter CHAPTER7 --- FUTURE STUDIES --- p.177 / Chapter CHAPTER8 --- REFERENCES --- p.180

MicroRNA（miRNAs）是一類細小的非編碼RNA（ncRNA），能透過轉錄後機制調節靶標基因的表達。miRNA的發現，不僅提出一個嶄新的基因調節機制，更強調了小ncRNA於不同的生理和發展過程中的重要性。最近的研究更進一步展示了miRNA失調與癌症發展之間的因果關係。 / 我們此前曾利用陣列分析，發現miRNA在肝細胞癌（HCC）中的失調模式，揭示了miR-145在HCC的普遍下調。在本論文的第一部分，定量逆轉錄聚合酶鏈反應（qRT-PCR）進一步證實了miR-145在50的肝細胞癌患者（n=80）的腫瘤中出現表達下調，而且miR-145的表達下調更與較短的无病生存期相關。其中一個低內源性miR-145的肝癌腫瘤樣本被建立為細胞株─HKCI-C2。此體外模型保持低miR-145水平，並於恢復miR-145表達後，抑製細胞存活和增殖。多個計算機演算法均預測了miR-145可針對胰島素樣生長因子（IGF）信號通路中的多個基因，包括胰島素受體底物（IRS1）-1，IRS2和胰島素樣生長因子1受體。這些假定目標的蛋白表達亦被miR-145下調。熒光素酶檢測進一步驗證了miR-145和IRS1/IRS2 3'-非編碼區的直接目標關聯。隨後的分析也確定miR-145能下調 IGF信號通路下游的信號傳導，即活性β-catenin水平。 / 最近出現的深度測序技術，為研究miRNome提供了一個前所未有的平台，以識別已知和新的miRNA。此外，現代生物信息學技術可同時對不同類型的小ncRNA，如PIWI-interacting RNA（piRNAs）進行分析。在本論文的第二部分中，我們利用Illumina大規模並行測序對兩個肝癌細胞株（HKCI-4和HKCI-8）和正常肝細胞株（MIHA）的小RNA轉錄組進行研究。生物信息學和生物功能分析揭示一種新型piRNA（取名為piR-Hep1）在肝腫瘤發生中的重要角色。在73例肝癌中，qRT-PCR結果顯示piR-Hep1在47的肝癌組織出現上調。PiR-Hep1的沉默能抑制肝癌細胞存活、遷移和侵襲，同時亦減少了Akt的磷酸化。在miRNA的分析中，miR-1323被發現在肝癌組織中大量表達，並與肝硬化背景下產生的肝腫瘤相關。此外，miR-1323出現過表達的肝硬化肝癌患者的無病和整體存活率亦較差（P<0.009）。 / 總觀來說，本論文首次發現miR-145可同時抑制引致肝癌的IGF信號通路中的多個傳導因子，亦突出了piR-Hep1的功能重要性和miR-1323在肝癌患者中的預後意義。此外，本研究表明，傳統的陣列分析和新一代的測序技術均能發現重要的miRNA。新一代測序技術對轉錄組的全面分析，將對研究各種不同類型的ncRNAs在肝癌發生發展過程中的參與提供新的思路。 / MicroRNAs (miRNAs) are a class of small non-coding RNAs (ncRNA) that post-transcriptionally regulate gene expression. The discovery of miRNAs not only puts forth an alternate gene regulatory mechanism, but also underscores the importance of small ncRNAs as pivotal regulators of diverse physiological and developmental processes. Recent studies have emphasized a causal link between miRNA deregulation and cancer development. / Our group has previously reported on dysregulated miRNA pattern in hepatocellular carcinoma (HCC) by array-based profiling, which revealed common downregulation of miR-145. In the first part of this thesis, quantitative reverse transcription polymerase chain reaction (qRT-PCR) corroborated reduced miR-145 expression in 50% of tumors in a cohort of 80 HCC patients, which also correlated reduced miR-145 expression with shorter disease-free survival of patients. One HCC tumor analyzed with low endogenous miR-145 was propagated as cell line. This in vitro model HKCI-C2 maintained low miR-145 level and upon restoration of miR-145 expression, a consistent inhibitory effect on cell viability and proliferation was readily observed. Multiple in silico algorithms predicted that miR-145 could target a number of genes along the insulin-like growth factor (IGF) signaling, including insulin receptor substrate (IRS1)-1, IRS2 and insulin-like growth factor 1 receptor. Protein expression of these putative targets was concordantly downregulated in the presence of miR-145. Luciferase reporter assay further verified direct target association of miR-145 to specific sites of IRS1 and IRS2 3’-untranslated regions. Subsequent analysis also affirmed the modulation of IGF signaling cascade by miR-145 as evident by reduction of the downstream mediator, namely, the active β-catenin level. / The recent advent of deep sequencing has provided an unprecedented platform to study the miRNome, in which both known and novel miRNAs can be identified. Moreover, bioinformatics advances have enabled different types of small ncRNAs, e.g. piwi-interacting RNAs (piRNAs), to be analyzed simultaneously. In the second part of this thesis, small RNA transcriptomes of two HCC cell lines (HKCI-4 and HKCI-8) and an immortalized hepatocyte line (MIHA) were examined using Illumina massively parallel sequencing. Combined bioinformatic and biological analyses revealed the involvement of a novel piRNA, designated as piR-Hep1, in liver tumorigenesis. piR-Hep1 was found to be up-regulated in 47% of HCC in a cohort of 73 HCC patients by qRT-PCR. Silencing of piR-Hep1 inhibited cell viability, motility and invasiveness with a concomitant reduction of Akt phosphorylation. In the analysis of miRNA, miR-1323 was found to be abundantly expressed in HCC and distinctly associated with tumors arising from a cirrhotic background. Furthermore, miR-1323 overexpression in cirrhotic-HCC correlated with poorer disease-free and overall survivals of patients (P<0.009). / Taken together, results from this thesis showed for the first time the pleiotropic effect of miR-145 on targeting multiple components of the oncogenic IGF signaling pathway in HCC. In addition, the functional importance of piR-Hep1 and the prognostic significance of miR-1323 in HCC were highlighted. Studies conducted demonstrated that important miRNAs can be discovered by both traditional array-based profiling and next-generation sequencing. Moreover, comprehensive definition of transcriptome by next-generation sequencing unveils virtually all types of ncRNAs and provides new insight into the liver carcinogenic events. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Law, Tak Yin. / "December 2012." / Thesis (Ph.D.)--Chinese University of Hong Kong, 2013. / Includes bibliographical references (leaves 180-200). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Abstract also in Chinese. / Abstracts also in Chinese. / Acknowledgements --- p.i / Publications --- p.ii / Abstract --- p.iii / 摘要 --- p.vi / Contents --- p.viii / List of Figures --- p.xiii / List of Tables --- p.xv / Abbreviations --- p.xvi / Chapter 1 --- Introduction --- p.1 / Chapter 1.1 --- Hepatocellular Carcinoma - One of the world’s most deadly killers --- p.2 / Chapter 1.2 --- MicroRNAs - a tiny molecule with enormous impacts --- p.10 / Chapter 1.2.1 --- Discovery of miRNAs --- p.11 / Chapter 1.2.2 --- Biogenesis and actions of miRNA --- p.13 / Chapter 1.3 --- MiRNAs and cancer --- p.16 / Chapter 1.4 --- Involvements of miRNAs in HCC etiological factors --- p.18 / Chapter 1.4.1 --- Viral hepatitis infection --- p.19 / Chapter 1.4.2 --- Chronic heavy alcohol consumption --- p.26 / Chapter 1.4.3 --- Dietary aflatoxin exposure --- p.28 / Chapter 1.4.4 --- Male gender --- p.31 / Chapter 1.4.5 --- Obesity --- p.33 / Chapter 1.5 --- Regulation of cancer-associated signaling network by microRNAs --- p.34 / Chapter 1.5.1 --- Apoptotic pathway --- p.37 / Chapter 1.5.1.1 --- Intrinsic pathway --- p.38 / Chapter 1.5.1.2 --- Extrinsic pathway --- p.39 / Chapter 1.5.2 --- Cell cycle regulators --- p.41 / Chapter 1.5.2.1 --- G₁/S transition --- p.42 / Chapter 1.5.2.2 --- G₂/M transition --- p.43 / Chapter 1.5.3 --- Receptor tyrosine kinase-mediated pathways --- p.45 / Chapter 1.5.3.1 --- c-MET-activated signaling --- p.45 / Chapter 1.5.3.2 --- PI3K-Akt --- p.47 / Chapter 1.5.3.3 --- RAS-RAF-MEK-ERK cascade --- p.48 / Chapter 1.5.4 --- TGF-ß signaling pathways --- p.50 / Chapter 1.5.5 --- Metastatic pathways --- p.52 / Chapter 1.5.5.1 --- MiRNAs with metastatic suppressing effects --- p.52 / Chapter 1.5.5.2 --- MiRNAs with metastatic promoting effects --- p.53 / Chapter 1.6 --- Clinical potentials of microRNAs - a killer or a cure? --- p.56 / Chapter 1.6.1 --- MiRNAs involvements in HCC risk prediction --- p.57 / Chapter 1.6.2 --- MiRNAs as diagnostic biomarkers --- p.59 / Chapter 1.6.3 --- MiRNAs as prognostic biomarkers --- p.60 / Chapter 1.6.4 --- Effects of miRNAs on responses to therapy --- p.61 / Chapter 1.7 --- Non-coding RNAs --- p.62 / Chapter 1.8 --- Aims of study --- p.63 / Chapter 2 --- Materials and Methods --- p.65 / Chapter 2.1 --- Quantitative reverse transcription polymerase chain reaction (qRT-PCR) --- p.66 / Chapter 2.1.1 --- Materials --- p.66 / Chapter 2.1.1.1 --- Total RNA extraction --- p.66 / Chapter 2.1.1.2 --- DNase treatment --- p.66 / Chapter 2.1.1.3 --- Reverse transcription --- p.66 / Chapter 2.1.1.4 --- Quantitative polymerase chain reaction --- p.66 / Chapter 2.1.2 --- Methods --- p.67 / Chapter 2.1.2.1 --- Total RNA extraction --- p.67 / Chapter 2.1.2.2 --- DNase treatment --- p.68 / Chapter 2.1.2.3 --- Reverse transcription --- p.69 / Chapter 2.1.2.4 --- Quantitative polymerase chain reaction --- p.69 / Chapter 2.2 --- Transfection --- p.70 / Chapter 2.2.1 --- Materials --- p.70 / Chapter 2.2.2 --- Methods --- p.70 / Chapter 2.2.2.1 --- Evaluation of HCC cells transfection efficiency --- p.70 / Chapter 2.2.2.2 --- Transfection --- p.71 / Chapter 2.3 --- In vitro functional assay --- p.72 / Chapter 2.3.1 --- Materials --- p.72 / Chapter 2.3.1.1 --- Cell viability assay --- p.72 / Chapter 2.3.1.2 --- Colony formation assay --- p.72 / Chapter 2.3.1.3 --- Cell cycle analysis --- p.72 / Chapter 2.3.1.4 --- Apoptosis assay --- p.72 / Chapter 2.3.1.5 --- Cell motility and invasion assay --- p.73 / Chapter 2.3.2 --- Methods --- p.73 / Chapter 2.3.2.1 --- Cell viability assay --- p.73 / Chapter 2.3.2.2 --- Colony formation assay --- p.74 / Chapter 2.3.2.3 --- Cell cycle analysis --- p.75 / Chapter 2.3.2.4 --- Apoptosis assay --- p.75 / Chapter 2.3.2.5 --- Cell motility and invasion assay --- p.76 / Chapter 2.4 --- Luciferase reporter assay --- p.78 / Chapter 2.4.1 --- Materials --- p.78 / Chapter 2.4.1.1 --- Cloning --- p.78 / Chapter 2.4.1.2 --- Cycle sequencing --- p.78 / Chapter 2.4.1.3 --- Luciferase reporter assay --- p.79 / Chapter 2.4.2 --- Methods --- p.79 / Chapter 2.4.1.1 --- Cloning --- p.79 / Chapter 2.4.2.2 --- Cycle sequencing --- p.81 / Chapter 2.4.2.3 --- Luciferase reporter assay --- p.82 / Chapter 2.5 --- Western blot --- p.84 / Chapter 2.5.1 --- Materials --- p.84 / Chapter 2.5.2 --- Methods --- p.85 / Chapter 2.5.2.1 --- Cell harvesting and protein quantitation --- p.86 / Chapter 2.5.2.2 --- Western blotting --- p.86 / Chapter 2.6 --- Small RNA Sequencing --- p.88 / Chapter 2.6.1 --- Materials --- p.88 / Chapter 2.6.2 --- Methods --- p.88 / Chapter 2.6.2.1 --- Sample preparation --- p.88 / Chapter 2.6.2.2 --- Cluster generation by bridge amplification --- p.88 / Chapter 2.6.2.3 --- Sequencing by synthesis --- p.89 / Chapter 2.7 --- Northern blot analysis --- p.94 / Chapter 2.7.1 --- Materials --- p.94 / Chapter 2.7.2 --- Methods --- p.94 / Chapter 2.7.2.1 --- Polyacrylamide gel electrophoresis (PAGE) --- p.94 / Chapter 2.7.2.2 --- Probe preparation --- p.95 / Chapter 2.7.2.3 --- Hybridization, stringency washes and signal detection --- p.95 / Chapter 3 --- Conventional miRNA profiling reveals miR-145 as a tumor suppressor in HCC --- p.97 / Chapter 3.1 --- Introduction --- p.98 / Chapter 3.2 --- Materials and Methods --- p.102 / Chapter 3.2.1 --- Patients --- p.102 / Chapter 3.2.2 --- qRT-PCR --- p.104 / Chapter 3.2.3 --- Cell line --- p.105 / Chapter 3.2.4 --- Transfection --- p.106 / Chapter 3.2.5 --- In vitro functional assay --- p.107 / Chapter 3.2.5.1 --- Cell viability assay --- p.107 / Chapter 3.2.5.2 --- Colony formation assay --- p.107 / Chapter 3.2.5.3 --- Flow cytometry assay --- p.107 / Chapter 3.2.6 --- miRNA target prediction --- p.109 / Chapter 3.2.7 --- Luciferase reporter assay --- p.110 / Chapter 3.2.8 --- Western blot --- p.112 / Chapter 3.2.9 --- Immunohistochemistry --- p.113 / Chapter 3.2.10 --- Statistical analysis --- p.114 / Chapter 3.3 --- Results --- p.115 / Chapter 3.3.1 --- Down-regulation of miR-145 in primary HCC --- p.115 / Chapter 3.3.2 --- Re-expression of miR-145 induced G₂-M arrest and apoptosis --- p.119 / Chapter 3.3.3 --- IRS1, IRS2 and IGF1R expressions --- p.124 / Chapter 3.3.4 --- miR-145 targeted both IRS1 and IRS2 and elicited IGF signaling --- p.126 / Chapter 3.4 --- Discussion --- p.131 / Chapter 4 --- Small RNA Deep sequencing reveals novel non-coding RNAs in HCC --- p.134 / Chapter 4.1 --- Introduction --- p.135 / Chapter 4.2 --- Materials and Methods --- p.136 / Chapter 4.2.1 --- Cell lines --- p.136 / Chapter 4.2.2 --- Patients --- p.137 / Chapter 4.2.3 --- Small RNA Sequencing --- p.139 / Chapter 4.2.4 --- Bioinformatics analysis --- p.140 / Chapter 4.2.4.1 --- Sequence mapping and ncRNA identification --- p.140 / Chapter 4.2.4.2 --- Putative miRNA prediction --- p.140 / Chapter 4.2.4.3 --- Putative piRNA identification --- p.140 / Chapter 4.2.4.4 --- Differentially-expressed ncRNAs identification --- p.141 / Chapter 4.2.5 --- qRT-PCR --- p.142 / Chapter 4.2.6 --- Northern blot analysis --- p.143 / Chapter 4.2.7 --- Transfection --- p.144 / Chapter 4.2.8 --- In vitro functional assays --- p.145 / Chapter 4.2.8.1 --- Cell viability assay --- p.145 / Chapter 4.2.8.2 --- Cell motility and invasion assay --- p.145 / Chapter 4.2.9 --- Western blot analysis --- p.147 / Chapter 4.2.10 --- Statistical analysis --- p.148 / Chapter 4.3 --- Results --- p.149 / Chapter 4.3.1 --- Small RNA Sequencing --- p.149 / Chapter 4.3.2 --- Up-regulation of putative piR-Hep1 in HCC --- p.155 / Chapter 4.3.3 --- piR-Hep1 silencing reduced cell viability and invasiveness --- p.159 / Chapter 4.3.4 --- Novel miR-1323 overexpression in HCC --- p.162 / Chapter 4.4 --- Discussion --- p.171 / Chapter 5 --- Concluding remarks and future perspectives --- p.175 / Chapter 6 --- References --- p.179

大腸癌是全世界最常見的癌症之一，亦是一個癌症死亡率的首要原因。大腸癌患者約50%在病程中會出現轉移病灶。近十年來，雖然多種被批准用於臨床治療的新化療藥顯著提高了大腸癌的治療效果，但是轉移性大腸癌病人的預後仍然很差。隨著各種分子生物技術的進步，新的治療標靶可能在大腸癌細胞株中被發現，並得以在病人標本中驗證。 / 在本研究中，我們採用即時定量多聚酶鏈反應(qPCR)陣列分析，比較大腸癌細胞株和正常大腸細胞株基因表達譜，試圖識別潛在的新的治療標靶。結果提示，與正常大腸細胞株 CCD-18Co 比較，Jagged 2 (JAG2) 和 Frizzled-3 (FZD3)基因 在大腸癌細胞株 SW480 和 SW620 中表達升高。病人大腸癌組織的免疫組織化學染色 (IS) 檢查進一步證實了上述結果，大腸癌組織較其癌旁正常組織表達3.1倍JAG2和6.6倍FZD3蛋白。因此， 我們假設JAG2和FZD3在大腸癌的發生中起重要作用。 / 為了檢驗該假設的真偽，我們運用RNA 干擾的方法進行功能缺失研究。通過該方法，大腸癌細胞株中JAG2 信使RNA和蛋白均能夠被下調，但是FZD3蛋白卻沒有顯示降低。為了弄清JAG2基因的功能，我們進行了單層細胞劃痕傷口癒合試驗和Matrigel 侵襲試驗。結果提示，JAG2 基因下調顯著抑制大腸癌細胞遷移和侵襲的能力。 / 為了調查參與上述功能的機制，我們利用腫瘤轉移相關基因的qPCR陣列分析，試圖檢測出JAG2基因敲除後上調或下調表達的轉移相關基因。結果顯示組織蛋白酶K (CTSK)，一種溶酶體半胱氨酸蛋白酶，在JAG2基因沉默的大腸癌細胞株中表達下調。為了闡明CTSK 活性在大腸癌細胞株侵襲能力中起到的作用，我們採用CTSK抑制劑處理大腸癌細胞株HCT116和DLD-1，發現這兩種細胞株的侵襲能力分別下降了36%和59%。總之, 這些發現表明CTSK可能是JAG2的下游靶基因，活性CTSK可能參與了JAG2介導的大腸癌細胞株侵襲能力。 / 以前的研究表明p38 MAPK通路參與癌細胞遷和侵襲能力的調控。通過Western blot方法，磷酸化的p38和磷酸化的STAT3被發現在JAG2基因沉默的大腸癌細胞中表達降低。p38抑制劑處理的 HCT116和DLD-1細胞降低了侵襲能力下降，同時遷移能力也由於p38抑制劑的處理而降低，支持p38可調控癌細胞遷移和侵襲能力的事實。 / 總之，我們的結果顯示JAG2高表達通過啟動CTSK和p38 MAPK通路，可能促進大腸癌轉移。因此，JAG2可能成為轉移性大腸癌治療的潛在標靶。 / Colorectal cancer (CRC) is one of the most frequent cancers worldwide and is a leading cause of cancer mortality. Around 50% of patients with CRC will experience metastases. Although significant progress has been made in CRC treatment within the last decade with the approval of multiple new chemotherapeutic agents, the prognosis for patients with metastatic CRC remains poor. With the advancement of molecular techniques, novel therapeutic targets are able to be discovered in CRC cell lines and validated in patient samples. / Therefore in this project, I aim to identify potential novel therapeutic targets by comparing the gene expression profile of colon cancer cell lines and a normal colon cell line using quantitative polymerase chain reaction (qPCR) arrays. Results showed that Jagged 2 (JAG2) and Frizzled-3 (FZD3) were up-regulated in the CRC cell lines SW480 and SW620 as compared to the normal colon cell line CCD-18Co. Those results were further validated by immunohistochemical staining (IS), which detected up-regulated JAG2 (3.1-fold) and FZD3 (6.6-fold) proteins expression in CRC tissues as compared to adjacent normal tissues. Thus I hypothesized that JAG2 and FZD3 may play an important role in CRC carcinogenesis. / In order to study the roles of FZD3 and JAG2 in CRC, loss-of-function studies by RNA interference (RNAi) were carried out. While the expression of FZD3 protein failed to be down-regulated by RNAi, JAG2 expression was successfully knocked down in CRC cell lines at both the mRNA and protein levels. Functional analyses using the monolayer scratch wound-healing assay and Matrigel invasion assay showed that JAG2 knockdown significantly inhibited migration and invasion in CRC cell lines. / To investigate the mechanisms involved, a tumour metastasis qPCR array was used to examine the changes in the expression level of metastasis-related genes after JAG2 gene knockdown. Results showed that the expression of Cathepsin K (CTSK), a lysosomal cystein protease, was found to be down-regulated in CRC cell lines following JAG2 silencing. To demonstrate the importance of CTSK activity in CRC cell invasion, HCT116 and DLD-1 CRC cell lines were treated with a CTSK inhibitor and its effect were assessed by the Matrigel invasion assay. Results showed that CTSK inhibition led to a 36% and 59% reduction in number of invaded cells in HCT116 and DLD-1 cell lines, respectively. Taken together, these findings show that CTSK may be a downstream target of JAG2 and that active CTSK may involve in JAG2 mediated invasion in CRC cell lines. / Previous works by others have shown that the p38 MAPK pathway is involved in the regulation of migration and invasive activity of cancer cell lines. Using Western blot analysis, the expression of phosphorylated p38 MAPK and phosphorylated STAT3 were found to be down-regulated following JAG2 depletion in CRC cell lines. In support of a role for p38 MAPK in the regulation of cancer cell migration and invasive capability, treatment with a p38 MAPK inhibitor was found to reduce the percentage of invasive cells and distance moved by migratory cells in HCT116 and DLD-1 cell lines. / In conclusion, my results show that JAG2 over-expression in CRC may promote cancer cell migration and invasion through activation of CTSK and the p38 MAPK pathway. Therefore, JAG2 may be a potential therapeutic target for treatment of metastatic CRC. / 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. / He, Wan. / Thesis (Ph.D.)--Chinese University of Hong Kong, 2013. / Includes bibliographical references (leaves 164-207). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Abstracts also in Chinese. / Abstract in English --- p.i / Abstract in Chinese --- p.iv / Acknowledgements --- p.vi / Chapter Chapter 1 --- Introduction --- p.1 / Chapter 1.1 --- Colorectal Cancer (CRC) --- p.1 / Chapter 1.1.1 --- Epidemiology and Incidence --- p.1 / Chapter 1.1.2 --- Histology --- p.2 / Chapter 1.1.3 --- Gender and Age --- p.4 / Chapter 1.1.4 --- Etiology of CRC --- p.4 / Chapter 1.1.4.1 --- Environment --- p.4 / Chapter 1.1.4.2 --- Hereditary Factors --- p.5 / Chapter 1.1.4.3 --- Dietary Factors --- p.6 / Chapter 1.1.4.4 --- Obesity --- p.6 / Chapter 1.1.4.5 --- Tobacco and alcoho --- p.7 / Chapter 1.1.4.6 --- Inflammatory bowel disease (IBC) --- p.7 / Chapter 1.1.5 --- Genetic Changes in CRC --- p.8 / Chapter 1.1.5.1 --- Chromosomal Aberration --- p.8 / Chapter 1.1.5.2 --- Tumor Suppressor Genes --- p.10 / Chapter 1.1.5.2.1 --- APC gene --- p.10 / Chapter 1.1.5.2.2 --- P53 gene --- p.11 / Chapter 1.1.5.2.3 --- SMAD4 gene --- p.11 / Chapter 1.1.5.3 --- Oncogenes --- p.12 / Chapter 1.1.5.3.1 --- Epidermal Growth Factor Receptor (EGFR) gene --- p.12 / Chapter 1.1.5.3.2 --- RAS gene and BRAF gene --- p.13 / Chapter 1.1.5.4 --- Proposed Two-hit Model for the Multistep Pathogenesis of CRC --- p.15 / Chapter 1.1.6 --- Clinical Presentation and Diagnosis --- p.16 / Chapter 1.1.7 --- Theatment --- p.16 / Chapter 1.1.7.1 --- Surgery --- p.16 / Chapter 1.1.7.2 --- Radiotherapy (RT) --- p.17 / Chapter 1.1.7.3 --- Concurrent Chemotherapy --- p.17 / Chapter 1.1.7.4 --- Target Therapy --- p.18 / Chapter 1.1.7.5 --- Colorectal Cancer Treatment by Stage --- p.19 / Chapter 1.1.7.6 --- Novel Strategies --- p.20 / Chapter 1.1.7.6.1 --- Epigenetic therapy --- p.20 / Chapter 1.1.7.6.2 --- Immunotherapy --- p.21 / Chapter 1.2 --- Pathways Involved in CRC Carcinogenesisand Progression --- p.22 / Chapter 1.2.1 --- Wnt Signaling Pathway --- p.22 / Chapter 1.2.2 --- Notch Signaling --- p.23 / Chapter 1.2.3 --- Nuclear Factor-kappa B (NF-κB) Signaling Pathway --- p.23 / Chapter 1.2.4 --- Phosphatidylinositol 3-kinase (PI3K) Signaling Pathway --- p.24 / Chapter 1.2.5 --- Crosstalk Among WNT, NOTCH, NF-κB and PI3K Signaling Pathway in CRC --- p.24 / Chapter 1.3 --- Hypothesis and Objectives of this Study --- p.28 / Chapter Chapter 2 --- Identification of Differentially Expressed Genes between Colorectal Cancer Cell Lines and A Normal Colon Cell Line --- p.29 / Chapter 2.1 --- Background --- p.29 / Chapter 2.2 --- Materials and Methods --- p.33 / Chapter 2.2.1 --- Cell Lines --- p.33 / Chapter 2.2.2 --- Identification of Differetially Expressed Genes by qPCR Arrays --- p.33 / Chapter 2.2.2.1 --- Total RNA Extraction --- p.33 / Chapter 2.2.2.2 --- RNA Quality Contol --- p.34 / Chapter 2.2.2.3 --- Reverse Transcription (RT) --- p.34 / Chapter 2.2.2.4 --- PCR Arrays --- p.34 / Chapter 2.3 --- Results --- p.36 / Chapter 2.3.1 --- Differentially Expressed Genes in WNT Signaling Pathway --- p.36 / Chapter 2.3.2 --- Differentially Expressed Genes in Notch Signaling Pathway --- p.40 / Chapter 2.3.3 --- Differentially Expressed Genes in NF-κB Signaling Pathway --- p.43 / Chapter 2.3.4 --- Differentially Expressed Genes in PI3K-AKT Signaling Pathway --- p.46 / Chapter 2.3.5 --- Choice of over-expressed genes for further validation and characterization --- p.49 / Chapter 2.4 --- Discussions --- p.53 / Chapter 2.4.1 --- WNT Signaling Pathway --- p.53 / Chapter 2.4.2 --- NOTCH Signaling Pathway --- p.54 / Chapter 2.4.3 --- NF-κB Signaling Pathway --- p.55 / Chapter 2.4.4 --- PI3K-AKT Signaling Pathway --- p.56 / Chapter 2.4.5 --- Choice of over-expressed genes for further validation and characterization --- p.56 / Chapter Chapter 3 --- JAG2, FZD3 and NOTCH4 Expression in Colorectal Cancer Cell Lines and Colorectal Cancer Tissues --- p.59 / Chapter 3.1 --- Background --- p.59 / Chapter 3.1.1 --- JAG2 Ligand --- p.59 / Chapter 3.1.2 --- FZD3 Receptor --- p.61 / Chapter 3.1.3 --- NOTCH4 Receptor --- p.62 / Chapter 3.2 --- Materials and Methods --- p.64 / Chapter 3.2.1 --- CRC Cell Lines --- p.65 / Chapter 3.2.2 --- CRC Tissues --- p.65 / Chapter 3.2.3 --- Quantitative RT-PCR --- p.66 / Chapter 3.2.4 --- Detection of JAG2, FZD3 and NOTCH4 Protein Expression in CRC Tissues by Immunohistochemical Staining (IS) --- p.67 / Chapter 3.2.5 --- Western Blot Assays --- p.68 / Chapter 3.2.5.1 --- Protein extraction --- p.68 / Chapter 3.2.5.2 --- SDS-PAGE gel electrophroresis --- p.68 / Chapter 3.2.5.3 --- Protein blotting --- p.68 / Chapter 3.2.6 --- Detection of JAG2 and FZD3 Protein Expression in CRC and Normal Colon Cell Lines by Western Blotting --- p.69 / Chapter 3.2.7 --- Statistical Analysis --- p.70 / Chapter 3.3 --- Results --- p.71 / Chapter 3.3.1 --- JAG2 and FZD3 but not NOTCH4 mRNA were Over -expressed in CRC Cell Lines --- p.71 / Chapter 3.3.2 --- Over-expression of JAG2 and FZD3 Proteins in CRC Tissues --- p.72 / Chapter 3.3.3 --- FZD3 Over-expression Correlated with Tumour-Node Metastasis (TNM) stages --- p.76 / Chapter 3.3.4 --- JAG2 and FZD3 Protein Expression in Colorectal Cancer and Normal Cell Lines --- p.77 / Chapter 3.4 --- Discussions --- p.78 / Chapter Chapter 4 --- Functional Analyses of JAG2 and FZD3 in CRC Cell Lines by RNA Interference --- p.81 / Chapter 4.1 --- Background --- p.81 / Chapter 4.2 --- Materials and Methods --- p.84 / Chapter 4.2.1 --- Transfection of siRNA into CRC Cell Lines --- p.84 / Chapter 4.2.2 --- Cell Proliferation Assay --- p.85 / Chapter 4.2.3 --- Monolayer Scratch Wound Healing Assay --- p.85 / Chapter 4.2.4 --- Matrigel Invasion Assay --- p.86 / Chapter 4.2.5 --- Statistical Analysis --- p.87 / Chapter 4.3 --- Results --- p.88 / Chapter 4.3.1 --- Knockdown of JAG2 and FZD3 Expression by RNA Interference --- p.88 / Chapter 4.3.2 --- Effect of JAG2 Knockdown on Migration of CRC Cell Lines --- p.91 / Chapter 4.3.3 --- JAG2 Knockdown by siRNA 2 Transfection Reduced Migratory Capability of HCT116, DLD-1and HT29 cell lines --- p.94 / Chapter 4.3.4 --- JAG2 Knockdown Impaired the Invasiveness of HCT116 and DLD-1 Cell Lines --- p.97 / Chapter 4.3.5 --- Decreased Migratory and Invasive Capabilities Induced by JAG2 Knockdown was not Due to Reduced Cell Proliferation --- p.100 / Chapter 4.4 --- Discussions --- p.102 / Chapter Chapter 5 --- NOTCH Pathway Inactivation by JAG2 Silencing Reduces Oncogenic Properties of HT29 but not HCT116 andDLD-1 CRC Cell Lines --- p.106 / Chapter 5.1 --- Background --- p.106 / Chapter 5.2 --- Materials and Methods --- p.109 / Chapter 5.2.1 --- CRC Cell lines --- p.109 / Chapter 5.2.2 --- Pharmacological Inhibition of NOTCH signaling by DAPT --- p.109 / Chapter 5.2.3 --- Combination of DAPT Treatment and JAG2 Silencing by siRNA --- p.109 / Chapter 5.2.4 --- Western Blotting --- p.109 / Chapter 5.2.5 --- Cell Proliferation Assay (MTS Assay) --- p.110 / Chapter 5.2.6 --- Monolayer Scratch Wound Healing Assay --- p.110 / Chapter 5.2.7 --- Matrigel Invasion Assay --- p.111 / Chapter 5.2.8 --- Statistical Analysis --- p.111 / Chapter 5.3 --- Results --- p.112 / Chapter 5.3.1 --- JAG2 Silencing Down-regulates Notch Pathway Signaling in CRC Cell Lines --- p.112 / Chapter 5.3.2 --- Inhibition of NOTCH Signaling by DAPT Treatment in CRC Cell Lines --- p.112 / Chapter 5.3.3 --- NOTCH Inhibition Does not Significantly Affect Cell Proliferation in CRC Cell Lines --- p.114 / Chapter 5.3.4 --- Suppression of NOTCH Signaling by DAPT Inhibits Migration in HT29 but not in HCT116 and DLD-1 CRC Cell Lines --- p.115 / Chapter 5.3.5 --- Suppression of NOTCH Signaling by DAPT does not Significantly Affect Invasiveness of HCT116 and DLD-1 CRC Cell Lines --- p.117 / Chapter 5.4 --- Discussions --- p.118 / Chapter Chapter 6 --- JAG2 Knockdown Inhibits Invasion in CRC Cell Lines through Inactivation of Cathepsin K --- p.121 / Chapter 6.1 --- Background --- p.121 / Chapter 6.2 --- Materials and Methods --- p.123 / Chapter 6.2.1 --- Human Tumour Metastasis RT2 Profiler[superscript TM] PCR Array --- p.123 / Chapter 6.2.2 --- Measurement of CTSK Gene expression level by Quantitative Real-Time PCR --- p.123 / Chapter 6.2.3 --- Immunohistochemical Staining (IS) of CTSK in CRC Tissues --- p.124 / Chapter 6.2.4 --- Pharmacological Inhibitior of CTSK in CRC Cell Lines --- p.124 / Chapter 6.2.5 --- Inhibition of CTSK in CRC Cell Lines for Migration Study --- p.124 / Chapter 6.2.6 --- Inhibition of CTSK in CRC Cell Lines for Invasion Study --- p.125 / Chapter 6.2.7 --- Western Blotting --- p.125 / Chapter 6.2.8 --- Statistical Analysis --- p.125 / Chapter 6.3 --- Results --- p.126 / Chapter 6.3.1 --- Identification of Metastasis Related Genes Which were Down-regulated by JAG2 Knockdown in HCT116 Cells --- p.126 / Chapter 6.3.2 --- Validation of Down-regulation of CTSK Gene by JAG2 Knockdown in HCT116 Cell Line by qRT-PCR --- p.126 / Chapter 6.3.3 --- JAG2 Knockdown Reduced Expression of Active CTSK Protein in CRC Cell Lines --- p.128 / Chapter 6.3.4 --- CTSK Protein Expression in CRC Tissue Samples --- p.130 / Chapter 6.3.5 --- Pharmacological Inhibition of CTSK Suppressed Invasiveness of CRC Cell Lines --- p.131 / Chapter 6.3.6 --- Pharmacological Inhibition of CTSK did not Affect Migration of CRC Cell Lines --- p.132 / Chapter 6.4 --- Discussions --- p.133 / Chapter Chapter 7 --- Depletion of JAG2 Inhibits Migration and Invasion in CRC Cell Lines through Inactivation of p38 MAPK/HSP27 Pathway --- p.137 / Chapter 7.1 --- Background --- p.137 / Chapter 7.2 --- Materials and Methods --- p.140 / Chapter 7.2.1 --- Pharmocological Inhibition of p38 MAPK Phosphorylation CRC Cell Lines --- p.140 / Chapter 7.2.2 --- Inhibition of p38 MAPK Phosphorylation for Migration Study in CRC Cell Lines --- p.140 / Chapter 7.2.3 --- Inhibition of p38 MAPK Phosphorylation for Invasion Study in CRC Cell Lines --- p.140 / Chapter 7.2.4 --- Knockdown of STAT3 by RNA interference --- p.141 / Chapter 7.2.5 --- Knockdown of STAT3 for Migration Study in CRC Cell Lines --- p.141 / Chapter 7.2.6 --- Knockdown of STAT3 for Invasion Study in CRC Cell Lines --- p.141 / Chapter 7.2.7 --- Western Blotting --- p.141 / Chapter 7.2.8 --- Statistical Analysis --- p.142 / Chapter 7.3 --- Results --- p.143 / Chapter 7.3.1 --- JAG2 Knockdown Inhibits p38 MAPK / HSP27 Pathway in CRC Cell Lines --- p.143 / Chapter 7.3.2 --- Inhibition of p38 MAPK / HSP27 Signaling Pathway Down-regulated Invasive Capability of CRC Cell Line --- p.145 / Chapter 7.3.3 --- Inhibition of p38 MAPK / HSP27 Signaling Pathway Down-regulated Migration of CRC Cell lines --- p.147 / Chapter 7.3.4 --- JAG2 Knockdown Inactivated p38 MAPK / HSP27 Pathway Independently of NOTCH Pathway in CRC Cell Lines --- p.149 / Chapter 7.3.5 --- JAG2 Knockdown Inhibits STAT3 Activation in CRC Cell Lines --- p.151 / Chapter 7.3.6 --- STAT3 Silencing Reduced Invasive Capability in CRC Cell Lines --- p.152 / Chapter 7.3.7 --- STAT3 Silencing Reduced Migratory Capability in CRC Cell Lines --- p.154 / Chapter 7.3.8 --- Inhibition of p38 MAPK Activity Suppressed STAT3 Activation in HCT116 Cells --- p.156 / Chapter 7.4 --- Discussions --- p.157 / Chapter Chapter 8 --- Conclusions and Future Works --- p.161 / Chapter 8.1 --- Conclusions --- p.161 / Chapter 8.2 --- Future work --- p.163 / References --- p.164 / Chapter Appendix 1 --- List of Figures and Tables --- p.208 / Chapter Appendix 2 --- Abbrevations used in this thesis --- p.212