京都大学 / 新制・課程博士 / 博士(医学) / 甲第25194号 / 医博第5080号 / 新制||医||1072(附属図書館) / 京都大学大学院医学研究科医学専攻 / (主査)教授 藤田 恭之, 教授 小濱 和貴, 教授 川口 義弥 / 学位規則第4条第1項該当 / Doctor of Agricultural Science / Kyoto University / DFAM

Liu, Xin. / "December 2009." / Thesis (M.Phil.)--Chinese University of Hong Kong, 2010. / Includes bibliographical references (leaves 119-126). / Abstracts in English and Chinese. / Abstract in English --- p.i / Abstract in Chinese --- p.iv / Acknowledgements --- p.vi / List of abbreviations --- p.vii / List of Tables List of Figures --- p.X xii / List of Publications --- p.xiv / Chapter Chapter 1 --- Literature Review --- p.1 / Chapter 1.1 --- Gastric cancer epidemiology and etiology --- p.1 / Chapter 1.2 --- Molecular carcinogenesis --- p.4 / Chapter 1.3 --- Tumor suppressor gene and the modes of tumor suppressor gene inactivation --- p.4 / Chapter 1.4 --- DNA methylation and carcinogenesis --- p.8 / Chapter 1.5 --- Identification of tumor suppressor genes --- p.15 / Chapter 1.6 --- "Vitamins, vitamin B complex, thiamine transporters and diseases" --- p.18 / Chapter 1.7 --- "Glucose metabolism, glycolysis and carcinogenesis" --- p.22 / Chapter 1.8 --- Clinical implications of DNA methylation --- p.28 / Chapter Chapter 2 --- Research Aim and Procedure --- p.31 / Chapter Chapter 3 --- Materials and Methods --- p.35 / Chapter 3.1 --- Cell lines and human tissue samples --- p.35 / Chapter 3.2 --- Cell culture --- p.35 / Chapter 3.3 --- Total RNA extraction --- p.36 / Chapter 3.4 --- Genomic DNA extraction --- p.37 / Chapter 3.5 --- Reverse transcription PCR (RT-PCR) --- p.38 / Chapter 3.5.1 --- Reverse transcription (RT) --- p.38 / Chapter 3.5.2 --- Semi-quantitative RT-PCR --- p.40 / Chapter 3.5.3 --- Real time RT-PCR --- p.42 / Chapter 3.6 --- General techniques --- p.44 / Chapter 3.6.1 --- DNA and RNA quantification --- p.44 / Chapter 3.6.2 --- Gel electrophoresis --- p.44 / Chapter 3.6.3 --- LB medium and LB plate preparation --- p.44 / Chapter 3.6.4 --- Plasmid DNA extraction --- p.45 / Chapter 3.6.4a --- Plasmid DNA mini extraction --- p.45 / Chapter 3.6.4b --- Plasmid DNA midi extraction --- p.46 / Chapter 3.6.5 --- DNA sequencing --- p.46 / Chapter 3.7 --- Methylation status analysis --- p.49 / Chapter 3.7.1 --- CpG island analysis --- p.49 / Chapter 3.7.2 --- Sodium bisulfite modification of DNA --- p.49 / Chapter 3.7.3 --- Methylation-specific PCR (MSP) --- p.50 / Chapter 3.7.4 --- Bisulfite genomic sequencing (BGS) --- p.53 / Chapter 3.8 --- Construction of expression plasmid DNA --- p.55 / Chapter 3.8.1 --- Construction of the SLC19A3-expressing vector --- p.55 / Chapter 3.8.2 --- Construction of the FBP1-expressing vector --- p.57 / Chapter 3.9 --- Functional analyses --- p.58 / Chapter 3.9.1 --- Monolayer colony formation assay --- p.58 / Chapter 3.9.2 --- Cancer cell growth curve analysis --- p.59 / Chapter 3.9.3 --- Lactate assay --- p.60 / Chapter 3.10 --- Statistical analysis --- p.61 / Chapter Chapter 4 --- Results --- p.62 / Chapter 4.1 --- Identification of novel candidate tumor suppressor genes downregulated by DNA methylation --- p.62 / Chapter 4.2 --- Selection of genes for further study --- p.62 / Chapter 4.3 --- Identification of SLC19A3 as a novel candidate tumor suppressor gene in gastric cancer --- p.64 / Chapter 4.3.1 --- Pharmacological restoration of SLC 19A3 downregulation in gastric cancer --- p.64 / Chapter 4.3.2 --- Methylation analysis of SLC 19A3 promoter region --- p.66 / Chapter 4.3.3 --- Functional analysis of SLC 19A3 in gastric cancer --- p.72 / Chapter 4.3.4 --- Clinicopathologic characteristics of SLC 19A3 promoter methylation in gastric cancer --- p.75 / Chapter 4.3.5 --- Discussion --- p.78 / Chapter 4.4 --- Identification of FBP1 as a novel candidate tumor suppressor gene regulated by NF-kB in gastric cancer --- p.85 / Chapter 4.4.1 --- Pharmacological restoration of FBP1 downregulation in gastric cancer --- p.85 / Chapter 4.4.2 --- Methylation analysis of FBP 1 promoter region --- p.87 / Chapter 4.4.3 --- Functional analysis of FBP 1 in gastric cancer --- p.93 / Chapter 4.4.4 --- Reduction of lactate generation under FBP1 expression --- p.95 / Chapter 4.4.5 --- Clinicopathologic characteristics of FBP 1 promoter methylation in gastric cancer --- p.98 / Chapter 4.4.6 --- NF-kB mediated FBP1 promoter hypermethylation in gastric cancer --- p.104 / Chapter 4.4.7 --- Discussion --- p.106 / Chapter Chapter 5 --- General discussion --- p.112 / Chapter Chapter 6 --- Summary --- p.117 / Reference list --- p.119

One of the major unresolved questions in cancer biology is why the majority of tumour cells express mutant p53 proteins. p53 is considered the prototype tumour suppressor protein, whose inactivation is the most frequent single genetic event in human cancer (Bourdon et al., 2005). Genetically-engineered p53-null knockout mice acquire multiple tumours very early on in life and human Li-Fraumeni families who carry germline mutations in p53 are highly cancer-prone (reviewed in Vousden and Lane, 2007). p53 mutant proteins have been found to acquire novel functions that promote cancer cell proliferation and survival, yet exactly why mutant p53s acquire oncogenic activity is still poorly understood. Mutant p53 has also been found to complex with wildtype p53, thus acting in a dominant negative way. However, this inhibition is incomplete since many cancers with mutant p53 alleles also have a loss of the second wild-type p53 allele and thus only express the mutant p53 (Baker et al., 1989). An N-terminal truncated p53 isoform, p47, arising from alternative splicing of the p53 gene (Ghosh et al., 2004) or by alternative initiation sites for translation (Yin et al. , 2002), has been described. Alternative splicing was found to be universal in all human multi-exon genes (Wang et al., 2008) and therefore determining the role of the p47 isoform with respect to the p53 gene is essential. Evidence in this study suggests that mutant p53 (p53RI75H) has a similar structure and function as p47, including the ability to complex with and impair both p53 and p73. Therefore, in addition to expressing a tumour suppressor protein, the p53 gene can also express an onco-protein (p47). This study therefore argues that tumours select for mutant p53 because it has gained the ability to function like p47, a wild-type p53 isoform.

The development of RNAi based technologies has given researchers the tools to interrogate processes as diverse as cancer biology, metabolism and organ development. Here I employ genome-wide shRNA screens to discover the genes involved in two different processes in carcinogenesis, oncogene-induced senescence [OIS] and epigenetic silencing of tumor suppressor genes [TSGs]. OIS is a poorly studied yet significant tumor suppressing mechanism in normal cells where they enter cell cycle arrest [senescence] or programmed cell death [apoptosis] in the presence of an activated oncogene. Here I employ a genomewide shRNA screen and identify a secreted protein, IGFBP7, that induces senescence and apoptosis in melanocytes upon introduction of the oncogene BRAFV600E. Expression of BRAFV600E in primary cells leads to synthesis and secretion of IGFBP7, which acts through autocrine/paracrine pathways to inhibit BRAF-MEK-ERK signaling and induce senescence and apoptosis. Apoptosis results from IGFBP7-mediated upregulation of BNIP3L, a proapoptotic BCL2 family protein. Recombinant IGFBP7 has potent pro-apoptotic and anti-tumor activity in mouse xenograft models using BRAFV600E-postive melanoma cell lines. Finally, IGFBP7 is epigenetically silenced in human melanoma samples suggesting IGFBP7 expression is a key barrier to melanoma formation. Next I investigated the factors involved in epigenetic silencing in cancer. The TSG p14ARFis inactivated in a wide range of cancers by promoter hypermethylation through unknown mechanisms. To discover p14ARF epigenetic silencing factors, I performed a genome-wide shRNA screen and identified ZNF304, a zinc finger transcription factor that contains a Krüppel-associated box [KRAB] repressor domain. I show that ZNF304 binds to the p14ARF promoter and recruits a KRAB co-repressor complex containing KAP1, SETDB1 and DNMT1 for silencing. We find oncogenic RAS signaling to promote the silencing of p14ARF by USP28-mediated stabilization of ZNF304. In addition I find ZNF304 to be overexpressed in human colorectal cancers and responsible for hypermethylation of over 50 TSGs known as Group 2 CIMP marker genes. My findings establish ZNF304 as a novel oncogene that directs epigenetic silencing and facilitates tumorigenicity in colorectal cancer.

No description available.

ARF, a potent tumor suppressor, positively regulates p53 by antagonizing MDM2, a negative regulator of p53, which in turn, results in either apoptosis or cell cycle arrest. ARF also suppresses the proliferation of cells lacking p53, and loss of ARF in p53-null mice, compared with ARF-null or p53-null mice, results in a broadened tumor spectrum and decreased tumor latency. This evidence suggests that ARF exerts both p53-dependent and p53-independent tumor suppressor activity. However, the molecular pathway and mechanism of ARF’s p53-independent tumor suppressor activity is not understood. The antiapoptotic, metabolically regulated, transcriptional corepressor C-terminal binding protein 2 (CtBP2) has been identified as a specific target of ARF’s p53-independent tumor suppression. CtBPs are phosphoproteins with PLDLS-binding motif and NADH-binding central dehydrogenase domains. ARF interacts with CtBP1 and CtBP2 both in vitro and in vivo, and induces their proteasome-mediated degradation, resulting in p53-independent apoptosis in colon cancer cells. ARF’s ability to target CtBP2 for degradation, and its induction of p53-independent apoptosis requires an intact interaction with CtBP2, and phosphorylation at S428 of CtBP2. As targets for inhibition by ARF, CtBPs are candidate oncogenes, and their expression is elevated in a majority of human colorectal adenocarcinomas specimens in comparison to normal adjacent tissue. Relevant to its targeting by ARF, there is an inverse correlation between ARF and CtBP expression, and CtBP2 is completely absent in a subset of colorectal adenocarcinomas that retains high levels of ARF protein. CtBPs are activated under conditions of metabolic stress, such as hypoxia, and they repress epithelial and proapoptotic genes. BH3-only genes such as Bik, Bim and Bmf have been identified as mediators of ARF-induced, CtBP2-mediated p53-indpendent apoptosis. CtBP2 repressed BH3-only genes in a tissue specific manner through BKLF (Basic kruppel like factor)-binding elements. ARF regulation of BH3-only genes also required intact interaction with CtBP2. ARF antagonism of CtBP repression of Bik and other BH3-only genes may play a critical role in ARF-induced p53-independent apoptosis, and in turn, tumor suppression. To study the physiologic effect of ARF/CtBP2 interaction at the organismal level, the p19ArfL46D knock-in mice, in which the Arf/CtBP2 interaction was abrogated, was generated. Analysis of the primary cells derived from these mice, revealed that the Arf/CtBP2 interaction contributes to regulation of cell growth and cell migration. Overexpression of CtBP in human tumors, and ARF antagonism of CtBP repression of BH3-only gene expression and CtBP-mediated cell migration may therefore play a critical role in the p53-independent tumor suppressor function/s of ARF.

Diss. (sammanfattning) Stockholm : Karolinska institutet, 2007. / Härtill 6 uppsatser.

Diss. (sammanfattning) Umeå : Univ., 2008. / Härtill 4 uppsatser.

Diss. (sammanfattning) Stockholm : Karolinska institutet, 2006. / Härtill 5 uppsatser.

Thesis (Ph.D.)--University of Alabama at Birmingham, 2008. / Title from first page of PDF file (viewed on June 26, 2009). Includes bibliographical references.