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

Dissertation (Ph.D.)--University of South Florida, 2008. / Includes vita. Also available online. Includes bibliographical references (leaves 93-111).

Diss. (sammanfattning) Linköping : Univ., 2002. / Härtill 4 uppsatser.

Diss. (sammanfattning) Linköping : Univ., 2004. / Härtill 4 uppsatser.

Diss. (sammanfattning) Stockholm : Karol. inst., 2003. / Härtill 5 uppsatser.

Diss. (sammanfattning) Stockholm : Karol. inst., 2006. / Härtill 4 uppsatser.

O câncer de mama é a principal causa de morte por câncer entre as mulheres no mundo. Fatores genéticos, comportamentais e ambientais afetam o risco de aparecimento dessa doença e seu desenvolvimento e progressão ocorrem pelo acúmulo de alterações genéticas/epigenéticas que levam a manutenção de sinais proliferativos nas células, fuga dos agentes supressores de crescimento e resistência à morte celular. O gene PHLDA1 (de Pleckstrin Homology-Like Domain, Family A, Member 1) codifica uma proteína de 401 aminoácidos que já foi descrita envolvida em distintos processos biológicos incluindo morte celular e, dessa forma, é frequentemente associada ao câncer. Perda progressiva de PHLDA1 já foi descrita em melanoma primário e metastático enquanto sua superexpressão foi descrita para tumores intestinais e pancreáticos. Em dados prévios de nosso grupo de pesquisa o gene PHLDA1 foi encontrado diferencialmente expresso em tumores de mama onde sua ausência estava relacionada com sobrevida livre de doença e sobrevida global reduzidas nas pacientes. Estudos do gene PHLDA1 em linhagens de mama são escassos e a compreensão de seu papel funcional e de como sua ausência pode estar relacionada com a redução da sobrevida em pacientes com câncer de mama permanecem obscuros. Com o objetivo de compreender a função de PHLDA1 em células epiteliais de mama, nós investigamos os efeitos da supressão do gene PHLDA1 em células MCF10A. A redução da expressão foi alcançada a partir de transfecção das células com vetores plasmidiais contendo shRNAs específicos para o transcrito de PHLDA1 e subsequentemente foram realizados ensaios funcionais. A expressão diminuida de PHLDA1 foi capaz de induzir acentuadas alterações morfológicas e comportamentais nas células MCF10A, incluindo mudança no padrão de ancoragem célula-célula e reorganização nos filamentos de actina, além de maior taxa de proliferação, migração e invasão das células. Além disso, em condições de baixa ancoragem, as células com expressão reduzida de PHLDA1 apresentaram mamosferas de formato irregular em comparação às células controle. Em conjunto, nossos resultados mostram que a diminuição da expressão de PHLDA1 em células MCF10A está relacionada a um comportamento agressivo e acentuadas alterações morfológicas. Estes dados são consistentes com atividade supressora tumoral de PHLDA1 em células epiteliais de mama / Breast cancer is the leading cause of cancer death among women worldwide. Genetic, behavioral and environmental factors affect the risk of onset of the disease. Breast cancer development and progression involves the accumulation of genetic/epigenetic changes that lead to maintenance of proliferative signals, evasion of growth suppressors and resistance to cell death. The PHLDA1 gene (Pleckstrin Homology-like domain, Family A, member 1) encodes a 401 amino acids protein that has been described involved in different biological processes including cell death and thus, is often associated with cancer. Progressive loss of PHLDA1 has been described in primary and metastatic melanoma while their overexpression has been reported for intestinal and pancreatic tumors. In previous data from our research group the PHLDA1 gene was found differentially expressed in breast tumors where its downregulation was related to shorter disease-free survival and overall survival of the patients. Literature regarding PHLDA1 in mammary epithelial cell lines is scarce and the understanding of their functional role and how its downregulation can be related to poor prognosis in breast cancer patients remain unclear. In order to understand the PHLDA1 function in breast epithelial cells, we investigated the effects of downregulation of PHLDA1 in MCF10A cells. The reduced expression was achieved from transfection of cells with plasmid vectors containing shRNAs for the specific transcript of PHLDA1 followed by functional assays. The decreased expression of PHLDA1 was sufficient to induce marked morphological and behavioral changes in MCF10A cells, including changes in cell-to-cell attachment pattern and actin reorganization, increased proliferation, migration and invasion rate of cells. Furthermore, in independent of attachment condition, cells with reduced expression of PHLDA1 formed mammospheras whit irregular shape compared to control cells. Taken together, our results showed that the decreased expression of PHLDA1 in MCF10A cells is related to aggressive behavior and marked morphological changes. These data are consistent with tumor suppressor activity for PHLDA1 in breast epithelial cells

O câncer de mama é o tumor mais incidente e a principal causa de mortalidade entre as mulheres no mundo. Não diferente de tantos outros tipos tumorais, o câncer de mama carrega em sua história uma etiologia complexa, heterogênea e multifatorial. O gene pró-apoptótico PAWR (PKC apoptosis WT1 regulator; também denominado como PAR-4, Prostate Apoptosis Response-4) é conhecido por induzir seletivamente apoptose em uma grande variedade de células de câncer. O papel de Par-4 como supressor tumoral vem sendo bem estabelecido nos últimos anos. Entretanto, pouco tem sido explorado sobre o papel e os mecanismos envolvendo a função supressora de Par-4 em câncer de mama. Em estudos prévios do nosso grupo, foi possível demonstrar que a expressão reduzida de Par-4 está associada a um pior prognóstico em câncer de mama e que esta proteína pode ter um papel importante na morfogênese da glândula mamária. Além disso, a investigação em células MCF-7 demostrou que a expressão de Par-4 aumenta a sensibilidade das células ao tratamento com docetaxel. Com objetivo de entender melhor o papel de Par-4 em células tumorais de mama, nós investigamos o efeito da supressão de Par-4 nas células MDA-MB-231 in vitro e in vivo. A redução da expressão foi alcançada a partir de transfecção das células com vetores plasmidiais contendo shRNAs específicos para o transcrito de Par-4 e subsequentemente foram realizados ensaios funcionais. A expressão reduzida de Par-4 foi capaz de aumentar a capacidade de formação de colônias das células MDA-MB-231 em cultura. Além disso, as células MDA-MB-231 transfectadas com shRNA-Par-4 tiveram uma redução significativa da morte celular (fase sub-G0\\G1 do ciclo celular), em particular da morte por apoptose (Anexina-FITC/PI), após o tratamento com diferentes quimioterápicos. Em nosso estudo, as células MDA-MB-231-Controle tratadas com docetaxel apresentaram aumento nos níveis de Par-4 secretado, o que não foi observado nas células MDA-MB-231-shPar-4. Finalmente, nossos resultados in vivo sugerem que a expressão diminuída de Par-4 pode modular o crescimento tumoral em camundongos Balb/c NUDE. Em conjunto, nossos dados colaboram com o papel supressor de Par-4 já descrito na literatura e confirmam sua ação supressora em diferentes linhagens de câncer de mama / Breast cancer is the most incident tumor and the leading cause of mortality among women worldwide. Like other forms of cancer, breast cancer has a complex, heterogeneous and multifactorial etiology. The pro-apoptotic gene PAWR (PKC apoptosis WT1 regulator; also known as PAR-4, Prostate Apoptosis Response-4) is known to selectively induce apoptosis in a wide variety of cancer cells. The role of Par- 4 as a tumor suppressor has been well established in recent years. However, little has been explored about the role and mechanisms of Par-4 suppressor function in breast cancer. Previous work from our research group demonstrated that the reduced expression of Par-4 is associated with a worse prognosis in breast cancer. Also an important role of Par-4 in the morphogenesis of the mammary gland was suggested. In addition, Par-4 overexpression in MCF-7 cells increased the sensitivity to docetaxel treatment. In order to better understand the role of Par-4 in breast tumor cells, we investigated the effect of Par-4 knock-down on MDA-MB-231 cells in vitro and in vivo. We performed shRNA-mediated Par-4 knockdown, and then carried out functional assays. The reduced expression of Par-4 was able to increase the colony formation capacity of MDA-MB-231 cells in culture. In addition, shRNA-Par-4 transfection in MDA-MB-231 cells led to a significant reduction of cell death (sub-G0/G1 cell cycle), particularly by apoptosis (Annexin-FITC/PI), after treatment with different chemotherapeutic agents. In our study, docetaxel-treated MDA-MB-231-Control cells showed increased levels of secreted Par-4, which was not observed in MDA-MB-231- shPar-4 cells. Finally, our in vivo results suggest that diminished Par-4 expression may modulate tumor growth in Balb/c NUDE mice. Taken together, our data support the suppressor role of Par-4 already described in the literature and confirm its suppressive action in different breast cancer cell lines

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.

Cancer is the leading cause of death in Hong Kong (21,300 new cases and 11,500 deaths in 2003), with nasopharyngeal carcinoma (NPC), esophageal cancer (ESCC), and colorectal cancer (CRC) among the common ones. For these tumors, most patients present with advanced stage disease and poor treatment outcome, with an urge of early detection. Epigenetic inactivation of tumor suppressor genes (TSG) by CpG methylation represents an important mechanism of tumorigenesis, in addition to genetic abnormalities. Tumor-specific methylation can also be used as biomarkers for the identification of novel TSGs and for cancer early diagnosis and prognosis prediction. / Finally, for the purpose of development of epigenetic biomarker for cancer molecular diagnosis, I screened gene methylation in the serum samples. Aberrant methylation of PCDH10 and DLC1 was detected in serum samples (2/14 (14%) and 4/14 (29%) respectively) from tumor patients but not in normal controls. It suggests that screening for PCDH10 and DLC1 methylation in sera could be a tumor-specific and non-invasive epigenetic biomarker for molecular diagnosis and prognostics. (Abstract shortened by UMI.) / In the second approach, 1-Mb array-based comparative genomic hybridization (aCGH) was carried out to detect DNA copy number aberrations, which contain potential TSG loci, in a panel of NPC and ESCC cell lines. Frequent deletions include: 1p36.3, 3p14-11, 4p16-15, 5p13-q12, 6p21-12, 8p22-cent, 9p, 9q22-31, 10p, 13q12, 14q32, 16q23-24, 17q11.2, 18q in NPC, and 1p21, 4q21, 7p21, 7q35, 8p22-23, 8q11, 10p11, 11q22, 13q31, 14q32, 18q11-23 in ESCC. Several deletions (3p14-11 and 16q23) were further investigated in detail in this study. More than 12 genes were identified to be frequently silenced by methylation in tumors, including FHIT (3p14), WNT5A (3p14), ADAMTS9 (3p14), FEZF2 (3p14), ROBO (3p12), CADM2 (3p12), EPHA3 (3p11), RAB (11q22), ADAMTS18 (16q23), and TUSC8 (16q23), while homozygous deletion of these genes was infrequently detected. Aberrant methylation of these genes was also frequently detected in primary tumors in a tumor-specific manner. The tumor suppressor functions of TUSC8, WNT5A, CADM2 and ROBO were further investigated and validated. Further experiment indicated that induction of tumor cell apoptosis may contribute to the tumor suppressor function of TUSC8. / Modified genomic methylation subtractive approaches using uracil-DNA glycosylase or combined with pharmacological demethylation were developed. GADD45G, PCDH10, ROR2, DLC1L1 were among a series of novel methylated targets identified by these approaches. Methylation-associated silencing of these genes was frequently detected in various types of tumor cell lines and primary tumors including NPC, ESCC and CRC, in a tumor-specific manner. Ectopic expression of these genes strongly suppressed tumor cell growth and colony formation of silenced tumor cells. Epigenetic inactivation of GADD45G is the major mechanism for the loss of its response to environmental stresses. Reintroduction of PCDH10 strongly suppressed tumor cell migration and invasion. Ectopic expression of DLC1L1 in silenced tumor cells resulted in a remarkable suppression of tumor cell clonogenicity, which depends on its GAP activity. Furthermore, DLC1L1, but not its inactivating mutants, inhibited Ras mediated oncogenic transformation. Thus, these identified genes are functional TSGs. / Ying Jianming. / "July 2007." / Adviser: Qian Tao. / Source: Dissertation Abstracts International, Volume: 69-01, Section: B, page: 0083. / Thesis (Ph.D.)--Chinese University of Hong Kong, 2007. / Includes bibliographical references (p. 147-173). / 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. / Abstract in English and Chinese. / School code: 1307.