Pancreatic adenocarcinoma (PDAC) is the fourth leading cause of cancer death in Western countries. The PDAC tumour microenvironment (TME) is characterized by a dense stromal reaction, consisting of many cell types including fibroblasts and immune cells. The chemokine receptor, CX3CR1 forms a high-affinity axis with its unique ligand CX3CL1 and is expressed on monocytes, macrophages and T cells. CX3CR1 is also present on pancreatic malignant cells, where it has been associated with metastasis formation. The aim of my project is to investigate the role of CX3CR1 in the progression and development of pancreatic cancer in a genetically engineered mouse model of PDAC, the CX3CR1GFP/GFPLSL-KRASG12D/+LSL-Trp53R172H/+Pdx1-Cre (CKPC) mouse. In these mice, the CX3CR1 protein is not functional but they express GFP. I have found that the absence of CX3CR1 in KPC mice has no effect in their lifespan and response to chemotherapy. Comparison of the immune infiltrate of the tumours revealed that the lack of CX3CR1 causes a significant decrease in T cells and a possible increase in myeloid cells in CKPC mice compared to KPC mice. Expression analysis of several inflammatory cytokines in the TME showed a significant difference in IL-10 between KPC and CKPC mice. There was also a significant increase in levels of, CX3CL1, both locally and in the plasma. Finally, we performed RNA-seq on KPC and CKPC tumours. My analysis revealed 607 differentially-expressed genes, some of which encoded other chemokines or protein regulating the immune system. In particular, I observed the upregulation of Cxcl10 and Cxcl12, and the downregulation of Gata3 and S100a4, which could explain the decrease in T cells in the TME of CKPC mice. In conclusion, although the lack of CX3CR1 modifies the TME in this genetic model of PDAC, these changes do not affect the lifespan or the response to chemotherapy.

Barrett's oesophagus (BO) is the major precursor of oesophageal adenocarcinoma (OA) and we do not understand the dynamics of the evolution of BO in order to identify patients at high risk of cancer. Studies have proposed that BO is a monoclonal lesion, however recent work has shown that there are multiple independent clones present. Project 1: Determines the evolution of polyclonal dysplasia through sequencing and mapping clones onto tissue sections. I show that several cases are polyclonal but in each case only one clone progresses to cancer, suggesting oesophageal cancers are monoclonal outgrows from polyclonal Barrett's dysplasia. Project 2: Aims to understand the clonal relationship between cells in glands displaying basal crypt dysplasia-like atypia (BCDA), as it is unclear whether those cells in the upper part of the gland arise from the same stem cell that generates the gland bases. Glands displaying BCDA show a common mutation between the dysplastic base and non-dysplastic surface suggesting a common cell of origin. Project 3: 50% of patients who undergo oesophagectomy for OA develop post-oesophagectomy Barrett's (neo- BO) within 3-5 years possibly due to a field effect, wherein pre-neoplastic cells remain post-resection in histologically normal areas of epithelium predisposing the patient to cancer recurrence. Here I show that no genetic link between the neo-BO and the cancer is present. Immunohistochemical analysis shows that neo- Barrett's glands are gastric in nature. Project 4: The stem cell dynamics and clonal expansion rates of BO are unknown. Here I employed diversity analysis of methylation patterns of CpG islands in the promoter regions of non-expressed genes as a molecular clock. My data suggests that 3-4 stem cells are found in each Barrett's gland. Methylation patterns within a gland were less diverse compared to adjacent and distant glands, suggesting BO is characterized by long periods of stasis followed by bursts of clonal expansions.

Introduction: The main risk factor for the development of oesophageal adenocarcinoma is Barrett’s oesophagus (BE). To diagnose those patients who will progress to cancer early to improve the dismal survival rate of oesophageal adenocarcinoma, patients with BE undergo regular endoscopic surveillance. The vast majority of patients, however, will never progress and are therefore monitored unnecessarily. Copy number changes have been shown to be important in the progression of BE to oesophageal adenocarcinoma (Li et al., 2014). Shallow whole genome sequencing (sWGS) has been established as a cost-effective method of investigating copy number changes in formalin fixed paraffin embedded (FFPE) tissue (Scheinin et al., 2014). We hypothesised that copy number alterations may be valuable markers in disease progression and aimed to characterise them in the progression of Barrett’s using sWGS in order to predict progression in patients from a point in time as close to baseline endoscopy as possible and to integrate p53 staining. Methods: To optimise sWGS we compared 50X WGS on frozen tissue with 0.1X WGS from FFPE tumour material from the same patient. To address poor cellularity in endoscopic biopsies, shallow WGS data from a 50% cellularity biopsy with a 90% frozen sample from a single patient were compared. Accounting for poor biopsy cellularity 0.4X coverage was used. We performed FFPE shallow WGS on 806 samples from an 89-patient cohort comprising a 1:1 ratio of patients who progressed to high grade dysplasia (HGD) and patients who never progressed. 1-31 samples per patient were collected over time and space throughout surveillance. Non-progressors had significantly longer follow-up (p-value = 0.0008). Data was processed based on published bioinformatic pipelines. Copy number analysis was carried out using a generalised linear model (GLM) in order to develop a predictive algorithm. Results: During optimisation, ˃85% of copy number changes were detected in both frozen and FFPE samples from spatially distinct regions of an individual tumour. We found 91% and 93% agreement in copy number calls using orthogonal platforms between 90% (frozen) and 50% (FFPE) cellularity samples from one tumour. In the 806 sample Barrett’s cohort, we observed larger copy number alterations in patients who progressed to cancer compared with non-progressors and significantly more CN alterations in progressor patients (p-value ˂ 0.001). More cancer-associated genes were affected in progressors and we observed significant heterogeneity between patients. There was also a greater level of complexity seen in the progressor patients when analysed using affinity propagation clustering. These data allowed us to develop a regression model to predict progression. Using the GLM model, we successfully classified samples as early as progressor or not with an AUC of 85.75% and a sensitivity and specificity of 84 and 79% respectively. At the patient level 94% progressor patients had at least one sample classified as at risk of progression and non-dysplastic progressor samples were classified as early as 13 years prior to HGD diagnosis. Depending on the classification threshold used, all samples over time and space were not classified as being at risk of progression in at least 60% patients who have not yet progressed to HGD/cancer. We observed 2 pathways to progression supporting previous observations. 90% of progressors had samples prior to their HGD or cancer diagnosis classified as being predisposed to progression suggestive of genetically unstable lesions from early on in surveillance that progressed to HGD over time. The remaining 10% appeared as non-progressors until their diagnosis of HGD. We investigated p53 expression in our patient cohort as the only biomarker to have successfully transitioned into the clinic for Barrett’s surveillance. Whilst we found our cohort to be representative in staining compared to other published cohorts, it did not contribute to the GLM and the copy number data out-performed the use of p53 IHC in the context of Barrett’s surveillance. Conclusions: We have optimised the use of shallow WGS in oesophageal adenocarcinoma and Barrett’s. Using these copy number data, we can confidently distinguish between patients who will progress to cancer and the majority of patients who will never progress. This approach has led to the development of a model for predicting progression in the clinical setting which is promising for further clinical validation.

子宮内膜癌是最常见的妇科恶性肿瘤，其中有80%属于子宮内膜腺样癌，这一癌症发病的分子机制尚未清楚。研究表明，癌基因表达异常或者功能异常在肿瘤的发生发展过程中具有重要的作用。另一方面，抑癌基因在肿瘤细胞中特异性甲基化失活通常导致细胞恶性转变和肿瘤的发生。本实验将阐明癌基因阴阳1和抑癌基因PCDH10在人类子宮内膜腺样癌发病中的作用。 / 本实验第一部分研究多功能转录因子阴阳1（YY1）在子宮内膜腺样癌发病过程中的作用。首先本实验证实YY1在子宮内膜腺样癌临床标本和癌细胞系中均明显表达上调，并且上调的程度与肿瘤的FIGO分期相关。接着体外细胞培养和裸鼠荷瘤模型的实验均提示抑制YY1 表达可抑制癌细胞增殖和体外迁移，而过表达YY1则促进癌细胞增殖。这些结果表明YY1在子宮内膜腺样癌发病中具有促进作用。进一步全细胞基因组转录谱分析提示YY1 介入子宮内膜腺样发病的各个方面，并通过抑制抑癌基因APC的表达发挥发挥重要作用。深入的分子机制研究发现一个新的表观抑制作用模型：YY1可募集EZH2等多梳蛋白到APC启动子区并导致后者组蛋白3赖氨酸27上三甲基化，从而抑制APC基因转录。此外，本实验还发现YY1在子宮内膜腺样癌的表达增高是由于微小RNA，miR-193a-5p，在此癌中表达下降所导致的。所以，本实验第一部分的结果揭示了miR-193a-5p-YY1-APC这条全新的信号通路在子宮内膜腺样癌发病中发挥重要作用，并可作为潜在的治疗靶点。 / 本实验第二部分鉴定出PCDH10作为子宮内膜腺样癌一个新的抑癌基因。通过5-氮杂-2'-氧胞嘧啶处理和亚硫酸氢钠测序的方法，我们证实抑癌基因PCDH10在子宮内膜腺样癌中失活是由于其启动子区DNA甲基化所致，并且这种DNA甲基化介导的PCDH10表达沉默在子宮内膜腺样癌临床标本和癌细胞系中很常见，但不存在于正常子宮内膜组织。另外，在子宮内膜腺样癌细胞系体外实验中恢复PCDH10的表达可抑制细胞增殖、单细胞克隆形成，促进细胞凋亡。 同时在体实验荷瘤模型中恢复PCDH10的表达也可抑制肿瘤细胞增殖，这些结果与其肿瘤抑制功能相符。 / 总之，本实验结果阐明了YY1和PCDH10在子宮内膜腺样癌发病过程中新的作用，拓展了子宮内膜腺样癌发病分子机制的研究并为其药物治疗提供了潜在的靶点。 / Endometrial cancer is the most common gynecologic malignancy and about 80% of these cancers are endometrial Endometrioid carcinoma (EEC). The molecular mechanisms underlying EEC tumorigenesis are under-explored. Aberrant expression and function of oncogenes promote tumor development by modulating many aspects of tumor cell growth. On the other hand, tumor specific promoter methylation on tumor suppressor genes (TSG), which are generally unmethylated in normal cells, usually initiate and promote malignant transformation and cancer initiation. Our study aims to characterize the functions of an oncogenic transcription factor Yin Yang 1 (YY1) and a novel tumor suppressor gene PCDH10 in Human Endometrioid Endometrial Adenocarcinoma. / In the first part of our study, we investigated the function of a multifunctional TF, YY1 in EEC tumorigenesis. We demonstrated YY1 is up-regulated in EEC cell lines and primary tumors and its expression is associated with FIGO stages. Depletion of YY1 inhibits EEC cell proliferation and migration both in vitro and in vivo whereas over-expression of YY1 promotes EEC cell growth. These results suggest that YY1 functions as an onocogenic factor in EEC. Transcriptome analysis revealed a significant effect of YY1 on critical aspects of EEC tumorigenesis and its down-regulation of APC transcripts. Further mechanistic investigation uncovered a new epigenetic silencing mode of Adenomatosis Polyposis Coli (APC) by YY1 through recruitment of EZH2 and trimethylation of histone 3 lysine 27 in its promoter region. Additionally, YY1 over-expression was found to be a consequence of miR-193a-5p down-regulation through direct miR-193a-5p-YY1 interplay. Our results therefore established a novel miR-193a-5p-YY1-APC regulatory axis contributing to EEC development, which may serve as future intervention target. / In the second part of our study, we identified PCDH10 as a novel tumor suppressor gene in EEC. By using bisulfate genomic sequencing combined with pharmacologic demethylation drug treatment, we elucidated that PCDH10 inactivation in EEC is a consequence of DNA hypermethylation on its promoter region. Further study suggested that hypermethylation-mediated PCDH10 silencing was a common event in EEC cell lines and clinical samples, but not in normal endometrial tissues. Restoration of PCDH10 expression in EEC cells suppressed cell proliferation, inhibited single cell colony formation and induced cell apoptosis; moreover, overexpression of PCDH10 inhibited EEC xenograft tumor growth in vivo.These results suggest PCDH10 acts as a tumor suppressor. / Together, our results reveal the novel functions of YY1 and PCDH10 in EEC. These findings add novel insights into the molecular mechanisms of EEC development and progression, which may serve as potential therapeutic targets for this disease. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Yang, Yihua. / Thesis (Ph.D.)--Chinese University of Hong Kong, 2012. / Includes bibliographical references (leaves 170-186). / Abstracts also in Chinese. / TITLE --- p.I / ABSTRACT --- p.III / ACKNOWLEDGEMENTS --- p.VII / PUBLICATION --- p.IX / ABBREVIATIONS --- p.X / LIST OF FIGURES --- p.XIII / LIST OF TABLES --- p.XVI / TABLES OF CONTENT --- p.XVII / Chapter Chapter 1 --- Introduction --- p.1 / Chapter 1.1 --- Endometrioid Endometrial Adenocarcinoma (EEC) --- p.1 / Chapter 1.1.1 --- Epidemiology --- p.2 / Chapter 1.1.2 --- Etiology and risk factors --- p.3 / Chapter 1.1.3 --- Treatment and prognosis --- p.8 / Chapter 1.1.4 --- Molecular Mechanisms --- p.9 / Chapter 1.1.5 --- APC and Wnt/β-catenin signaling pathway --- p.12 / Chapter 1.1.6 --- Summary --- p.18 / Chapter 1.2 --- Epigenetic modifications and EEC --- p.20 / Chapter 1.2.1 --- Epigenetic modifications --- p.20 / Chapter 1.2.2 --- Epigenetic and cancer --- p.21 / Chapter 1.2.5 --- Summary --- p.30 / Chapter Chapter 2 --- Material and Method --- p.31 / Chapter 2.1 --- Tissue samples --- p.31 / Chapter 2.2 --- Cell culture --- p.32 / Chapter 2.3 --- Cell proliferation assays --- p.33 / Chapter 2.4 --- Cell migration assay --- p.34 / Chapter 2.5 --- 3-deazaneplanocin A (Dznep) or 5-aza-2'-deoxycytidine (5-aza) treatment --- p.34 / Chapter 2.6 --- Computational prediction --- p.35 / Chapter 2.7 --- Cell cycle assay --- p.35 / Chapter 2.8 --- Apoptosis assay --- p.36 / Chapter 2.9 --- Total RNAs, Total proteins and Genomic DNA extraction --- p.36 / Chapter 2.10 --- Bisulfite Genomic Sequencing --- p.38 / Chapter 2.11 --- Oligonucleotides --- p.39 / Chapter 2.12 --- RT-PCR, Semi-quantitative PCR and Real-time PCR --- p.41 / Chapter 2.13 --- microRNA validation --- p.43 / Chapter 2.14 --- Plasmid construction --- p.43 / Chapter 2.15 --- Transfection --- p.45 / Chapter 2.16 --- Luciferase reporter assay --- p.45 / Chapter 2.17 --- Western blotting --- p.46 / Chapter 2.18 --- Immunofluorescence ( IF ) --- p.48 / Chapter 2.19 --- Immunohistochemistry (IHC) --- p.50 / Chapter 2.20 --- ChIP assay --- p.53 / Chapter 2.21 --- Sequencing and base calling --- p.55 / Chapter 2.22 --- Read mapping to genome with splice-aware aligner sequenced --- p.55 / Chapter 2.23 --- Xenograft mouse model --- p.55 / Chapter 2.24 --- Statistical analysis --- p.57 / Chapter Chapter 3 --- Yin Yang 1 Plays an Oncogenic Role in Human Endometrioid Endometrial Adenocarcinoma --- p.58 / Chapter 3.1 --- YIN YANG 1(YY1) --- p.58 / Chapter 3.1.1 --- YY1 structure --- p.58 / Chapter 3.1.2 --- YY1 function --- p.59 / Chapter 3.1.3 --- YY1 and epigenetic --- p.61 / Chapter 3.1.4 --- YY1 and cancer --- p.62 / Chapter 3.1.5 --- Regulation of YY1 expression and activity --- p.66 / Chapter 3.2 --- Results --- p.68 / Chapter 3.2.1 --- YY1 is up-regulated in EEC lines and localizes in nuclei of EEC cells --- p.68 / Chapter 3.2.2 --- YY1 expression level is associated with EEC clinicopathological features --- p.72 / Chapter 3.2.3 --- Knock-down of YY1 by RNAi inhibits EEC cell proliferation and migration --- p.77 / Chapter 3.2.4 --- Ectopic expression of YY1 promotes EEC cell proliferation --- p.84 / Chapter 3.2.5 --- YY1 does not affect EEC cell cycle and cell apoptosis --- p.91 / Chapter 3.2.6 --- Genome-wide characterization of YY1-mediated transcriptome changes --- p.94 / Chapter 3.2.7 --- Gene Ontology analysis of YY1 targets on EEC tumorigenesis --- p.98 / Chapter 3.2.8 --- YY1 inhibits APC gene expression and functions --- p.101 / Chapter 3.2.9 --- YY1 inhibits APC expression through recruiting EZH2 and causing H3K27me3. --- p.105 / Chapter 3.2.10 --- Knock-down of YY1 does not change DNA methylation status of CpG island of APC gene --- p.117 / Chapter 3.2.11 --- SiYY1 oligo injection inhibits tumor grows in vivo --- p.119 / Chapter 3.2.12 --- miR-193a-5p is down-regulated in EEC cell lines and clinical samples --- p.126 / Chapter 3.2.13 --- miR-193a-5p targets YY1 3’UTR and inhibits YY1 expression --- p.128 / Chapter 3.2.14 --- miR-193a-5p inhibits tumor grow in vivo --- p.133 / Chapter 3.3 --- Discussion --- p.136 / Chapter 3.3.1 --- YY1 oncogenic functions in EEC --- p.136 / Chapter 3.3.2 --- YY1 epigenetically silences APC --- p.138 / Chapter 3.3.3 --- miR-193a-5p down-regulates YY1 in EEC --- p.139 / Chapter 3.4 --- Conclusion --- p.141 / Chapter Chapter 4 --- The tumor suppressive functions of PCDH10 in Human Endometrioid Endometrial Adenocarcinoma --- p.143 / Chapter 4.1 --- Introduction --- p.143 / Chapter 4.1.1 --- PCDH10 structure and function --- p.143 / Chapter 4.1.2 --- PCDH10 and tumor --- p.146 / Chapter 4.1 --- Results --- p.149 / Chapter 4.2.1 --- PCDH10 is down-regulated in EEC cell lines and clinical samples --- p.149 / Chapter 4.2.2 --- PCDH10 is hypermethylated in EEC cell lines and clinical samples --- p.150 / Chapter 4.2.3 --- Pharmacologic demethylation restores PCDH10 expression in EEC cell lines --- p.152 / Chapter 4.2.4 --- Ectopic over-expression of PCDH10 inhibits EEC cell proliferation --- p.154 / Chapter 4.2.5 --- PCDH10 over-expression induces EEC cell apoptosis --- p.161 / Chapter 4.2.6 --- PCDH10 over-expression inhibits tumor grows in vivo --- p.166 / Chapter 4.3 --- Discussion and future plan --- p.169 / REFERENCE --- p.170

Made available in DSpace on 2014-06-11T19:31:04Z (GMT). No. of bitstreams: 0 Previous issue date: 2008-02-22Bitstream added on 2014-06-13T19:40:52Z : No. of bitstreams: 1 conde_sj_dr_botfm.pdf: 1083233 bytes, checksum: a328bfb8396940e543c7e1865edd1942 (MD5) / Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) / Sabe-se que o E2 e o status hormonal da paciente são importantes para a proliferação e o tratamento do câncer de mama, quanto ao T3, apesar dos estudos epidemiológicos serem ainda contraditórios em relação a sua influência no câncer de mama, estudos laboratoriais demonstram sua capacidade de induzir a sua proliferação e induzir genes que são induzidos por estrógeno. Portanto a hipótese formulada foi que os hormônios tireoidianos modulam a expressão dos mesmos genes que o estrógeno em carcinoma de mama. Para materializar a hipótese o objetivo foi comparar a expressão dos genes TGFA, TGFB1 e PGR, resultantes da ação do estrógeno, e dos genes TNFRSF9, BMP6 e THRA, resultantes da ação do hormônio tireoidiano, combinados ou não com a ação do Tamoxifem, em carcinoma de mama colocado em cultura primária. Foram selecionadas 12 pacientes do sexo feminino, menopausadas, com Carcinoma de Mama estádio grau 1 e 2 , do Hospital do Câncer de São Paulo, verificou-se a existência de disfunção tireoidiana concomitante, através das dosagens laboratoriais de T3L, T4L, TSH, TPO-AB, assim como o status hormonal em relação aos níveis estrogênicos. Fragmentos tumorais obtidos cirurgicamente foram colocados em cultura primária. Essas células foram tratadas com E2 (10-7M), T3 (10-8M), E2 (10-7M) + TAM (10-6M), T3 (10-8M) + TAM (10-6M), TAM (10-6M), com posterior extração de RNA total. Foi utilizada a metodologia de PCR em Tempo Real para verificar a ação do T3 e do E2 em genes normalmente induzidos pelo E2 (PGR, TGFA e TGFB1), verificada a expressão de genes que aumentam (TNFRSF9 e THRA) e diminuem (BMP6) pela ação do T3 em seu receptor (TR) e a presença de mutações no TR. Não foram encontradas mutações envolvendo o receptor de hormônio... / It is known that E2 and the patient’s hormonal status is important for the proliferation and the treatment of the breast cancer. Despite the contradictory epidemic studies of T3 and its influence in the breast cancer, laboratory studies demonstrate its capacity to induce not only its proliferation but also genes that are induced by estrogen. Therefore the hypothesis was that the thyroid hormones modulate the expression of the same genes as the estrogen in breast carcinoma. The objective was to compare the expression of the genes TGFA, TGFB1 and PGR, resultant of the estrogen action and genes TNFRSF9, BMP6 and THRA, resultant of the thyroid hormone action, combined or not with the Tamoxifen action in breast carcinoma in primary culture. 12 menopausal patients with breast carcinoma stages 1 and 2, of São Paulo Cancer Hospital were selected. The existence of concomitant thyroid dysfunction was verified through laboratory dosages of T3L, T4L, TSH, TPOAB, as well as the hormonal status in relation to the estrogen levels. Tumor fragments surgically obtained were put in primary culture. Those cells were treated with E2 (10-7M), T3 (10-8M), E2 (10-7M) + …, with subsequent extraction of total RNA. The methodology of PCR was used in Real Time to verify the action of T3 and E2 in genes usually induced by E2 (PGR, TGFA and TGFB1), verified the expression of genes which increase (TNFRSF9 and THRA) and decrease (BMP6) by the action of T3 in its receptor (TR) and the presence of mutations in TR. Mutations involving the thyroid hormone receptor were not found. Related to the genic expressions, in the ER positive patients, the treatment with T3 increased the expression of TGFA, THRA, TNFRSF9 and PGR genes significantly and reduced the expression of TGFB1 and BMP6. When... (Complete abstract click electronic access below)

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This study was designed to determine the distribution of fatty acids and cholesterol in total tumor and mammary tissues. Fatty acid profiles of phospholipid fractions from tumors and mammary glands were also determined. Fatty acids and cholesterols were analyzed by gas liquid chromatography. Methodology was developed for phospholipid separation by high performance liquid chromatography.Tumors derived from mammary glands in Strain A/ST mice were found to contain two to three times the amount of cholesterol compared to normal mammary glands. Mammary glands from safflower fed mice contained significantly higher percentages of linoleic acid. Linoleic acid content in stearicacid (SA-1) fed mice was sharply reduced. linoleic acid in mammary glands of animals fed a high fat Stearic acid (SA-4) , corn oil and stock diet fed animals displayed similar fatty acid profiles. Fatty acid analysis of tumors excised from mice fed the experimental diets showed similar patterns in comparison to normal mammary glands. The similar distributions were in the 18 carbon fatty acids. Distributions of phospholipid fatty acids in tumors and mammary glands were similar. Mammary gland phospholipids displayed increased percentages of short chain (14 carbons and under) fatty acids. Results of these studies demonstrate an increased availability of diet rich in polyunsaturated fatty acids.