published_or_final_version / Physiology / Master / Master of Philosophy

There is some evidence to suggest that the pineal gland influences neoplastic growth. Either crude or partially purified pineal extracts have been used to treat malignant neoplasms in humans. More compelling evidence indicates that the pineal hormone melatonin, in addition to its well-known antireproductive effects, may also exert oncostatic effects particularly in animal models of human breast cancer. The purpose of this study was to examine a possible direct action of melatonin on the growth morphology and physiology of human breast cancer cells in vitro. Studies are described in which physiological concentrations of melatonin are shown to have markedly inhibitory effects directly on MCF-7 human breast cancer cells grown in culture. This antimitotic effect is not observed in MCF-7 cells at supra- or subphysiological concentrations of melatonin. This growth-inhibitory effect appears to be tissue specific since fibroblastic cells were not affected by melatonin. Other pineal indoles failed to inhibit the proliferation of this human breast cancer cell line, suggesting that this growth-inhibitory effect is specific for melatonin and is not a general characteristic shared among the family of pineal indoles. Reductions in media serum concentrations dramatically suppressed the response of cells to melatonin's inhibitory action. Serum values of 2.5 percent or lower resulted in a loss of melatonin's action as did growing the cells in serum-free medium or medium containing charcoal-treated serum. It appears that certain serum factors are necessary for these cells to respond to melatonin's antiproliferative action. Melatonin, when added to cells grown in media supplemented with 10 percent fetal calf serum decreased the synthesis of proteins and resulted in morphological alterations suggestive of a sublethal toxic injury. Melatonin appears to have a direct role in inhibiting the proliferation of breast cancer cells; however, the presence of melatonin per se does not seem to be the fundamental cause of this antimitotic action since no activity is observed when cells are propagated in media containing charcoal-treated fetal calf serum or serum-free medium. There appears to be a requirement for certain serum factors in this antiproliferative action. Two factors that have proved important in this process are the hormones estradiol and prolactin. (Abstract shortened with permission of author.)

鈣粘蛋白是一類通過影響細胞粘附和細胞信號通路在腫瘤發生中起重要作用的細胞間粘附分子。鈣粘蛋白超家族包括經典鈣粘蛋白和非經典鈣粘蛋白，其中非經典鈣粘蛋白包含了原鈣粘蛋白。啟動子CpG甲基化調控下的基因沉默或表達下調是腫瘤發生中一個關鍵事件的觀點現已得到廣泛認可。一些鈣粘蛋白家族成員，如鈣粘蛋白-1/4/13（CDH1，CDH4，CDH13）被已有研究報導是受擬遺傳學調控沉默的功能性腫瘤抑制基因。本研究主要針對兩個鈣粘蛋白家族成員鈣粘蛋白-11（CDH11）和原鈣粘蛋白-10（PCDH10）進行腫瘤發生相關功能和機制的研究。 / CDH11位於雜合性缺失（LOH）經常發生而預示可能存在抑癌基因的染色體16q21-22區域，我們實驗室先前通過基因組芯片雜交技術（aCGH）對腫瘤細胞系的研究已發現它是該區域一個可能的抑癌基因。我們通過進一步的半定量反轉錄聚合酶鏈反應（RT-PCR）發現CDH11在正常組織和永生化正常上皮細胞中廣泛表達，但在各腫瘤細胞系中表達下降。甲基化特異性聚合酶鏈反應（MSP）和亞硫酸氫鹽處理的基因組測序（BGS）檢測到CDH11啟動子甲基化常發生于腫瘤細胞和腫瘤組織中。在CDH11表達缺失的腫瘤細胞中重新導入該基因的表達可顯著減少細胞克隆的形成，誘導細胞凋亡并抑制腫瘤細胞的遷移。通過更深入的機制研究，我們發現CDH11通過抑制Wnt/β-catenin信號通路發揮功能。 / 本研究的另一個鈣粘蛋白家族成員是PCDH10。之前我們實驗室的工作已經證實了PCDH10是一個在鼻咽癌和食管癌中受啟動子甲基化調控的抑癌基因,這裡我們主要研究它在大腸癌發病中的功能和機制。我們發現在PCDH10表達缺失的大腸癌細胞中重新導入PCDH10表達可顯著抑制腫瘤細胞的克隆形成，細胞遷移和幹細胞性。機制研究顯示PCDH10抑制Wnt/β-catenin和RhoA信號轉導通路，并進一步抑制腫瘤上皮細胞-間充質轉化（EMT）的過程，誘導幹細胞標記的下調。 / 綜上所述，本研究顯示CDH11和PCDH10兩個鈣粘蛋白家族成員在多種腫瘤中廣泛受甲基化調控失活，它們是重要的Wnt/β-catenin信號通路拮抗因素，可抑制腫瘤細胞的克隆形成和細胞遷移 / Cadherins are an important group of cell-cell adhesion molecules, which play crucial roles in tumorigenesis by affecting cell adhesion and cell signaling. Cadherin superfamily consists of classical cadherins and non-classical cadherins including protocadherins. It has been well recognized that silencing or downregulation of tumor suppressor genes (TSGs) by promoter CpG methylation is a critical event in human tumorigenesis. Some cadherin family members, such as CDH1, CDH4, CDH13, have been reported as functional TSGs silenced through epigenetic regulation. In this study, I mainly focus on the function and mechanism studies of two cadherin members-Cadherin 11(CDH11) and Protocadherin 10 (PCDH10). / CDH11 is located in 16q21-22, a region with frequent loss of heterozygosity (LOH), indicating the presence of candidate TSG. Previously, our lab also identified CDH11 as a candidate TSG through array-CGH of tumor cell lines. I further found by semi-quantitative RT-PCR that CDH11 was broadly expressed in normal tissues while frequently downregulated in multiple tumor cell lines, but not in immortalized normal epithelial cells. Methylation-specific PCR (MSP) and bisulfite genomic sequencing (BGS) detected frequent promoter methylation of CDH11 in tumor cell lines and primary tumor samples. Ectopic expression of CDH11 dramatically reduced tumor cell clonogenecity, induced tumor cell apoptosis and inhibited tumor cell migration. By further mechanism study, I found that CDH11 is a negative inhibitor of Wnt/β-catenin signaling pathway. / Another cadherin family protein which I chose to study is PCDH10. Previously our lab identified PCDH10 as a TSG by promoter methylation in nasopharyngeal and esophageal carcinomas. I studied the function and mechanism of PCDH10 in the pathogenesis of colon cancer. I found ectopic expression of PCDH10 strongly suppressed colon tumor cell clonogenecity, migration and stemness. Moreover, I found that PCDH10 repressed Wnt/β-catenin and RhoA signaling, thus further inhibited the epithelial-to-mesenchymal transition (EMT) of tumor cells and downregulated stem cell markers. / In summary, this study demonstrates two cadherin family members CDH11 and PCDH10, as important antagonists to Wnt/β-catenin signaling pathway, suppress tumor cell clonogenecity, migration, and are also frequently inactivated by epigenetic mechanism in multiple tumors. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Zhang, Yanjiao. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2012. / Includes bibliographical references (leaves 84-95). / Abstracts also in Chinese. / Abstract --- p.i / Acknowledgements --- p.iii / Table of Contents --- p.vi / List of Tables --- p.x / List of Figures --- p.xi / List of Abbreviations --- p.xiii / List of Publications --- p.xvi / Chapter Chapter 1 --- Introduction and Literature Review --- p.1 / Chapter 1.1 --- Cancer --- p.1 / Chapter 1.1.1 --- General introduction about cancer --- p.1 / Chapter 1.1.2 --- Oncogenes and TSGs --- p.3 / Chapter 1.1.3 --- Cancer mechanism models --- p.3 / Chapter 1.2 --- Cancer Epigenetics --- p.5 / Chapter 1.2.1 --- DNA methylation --- p.6 / Chapter 1.2.2 --- DNA methylation and gene silencing --- p.7 / Chapter 1.2.3 --- DNA methylation and cancer --- p.7 / Chapter 1.2.4 --- Clinical implications of DNA methylation --- p.8 / Chapter 1.3 --- Cadherins --- p.10 / Chapter 1.3.1 --- General introduction of cadherin superfamily --- p.10 / Chapter 1.3.2 --- Cadherin classification --- p.10 / Chapter 1.3.3 --- Cadherin and cancers --- p.12 / Chapter 1.3.4 --- Cadherin switch and EMT in cancer --- p.16 / Chapter 1.4 --- Wnt/β-catenin signaling pathway and cancer --- p.16 / Chapter 1.4.1 --- Wnt/β-catenin signaling pathway --- p.16 / Chapter 1.4.2 --- Wnt/β-catenin signaling pathway in cancer --- p.18 / Chapter 1.4.3 --- Epigenetic silencing of Wnt/β-catenin signaling --- p.20 / Chapter 1.4.4 --- Wnt/β-catenin signaling pathway in CRC --- p.21 / Chapter Chapter 2 --- Aims of the Study --- p.22 / Chapter Chapter 3 --- Materials and Methods --- p.25 / Chapter 3.1 --- Cell lines and tissue samples --- p.25 / Chapter 3.1.1 --- Cell lines --- p.25 / Chapter 3.1.2 --- Maintenance of cell lines --- p.25 / Chapter 3.1.3 --- Drug treatment of cell lines --- p.26 / Chapter 3.1.4 --- Normal and primary tissues --- p.26 / Chapter 3.1.5 --- Total RNA extraction --- p.27 / Chapter 3.1.6 --- Genomic DNA extraction --- p.28 / Chapter 3.2 --- Gene expression analysis --- p.29 / Chapter 3.2.1 --- Reverse transcription (RT) --- p.29 / Chapter 3.2.2 --- Semi-quantitative RT-PCR --- p.30 / Chapter 3.3 --- Methylation Analysis --- p.32 / Chapter 3.3.1 --- CpG island prediction --- p.32 / Chapter 3.3.2 --- Sodium bisulfite treatment of genomic DNA --- p.33 / Chapter 3.3.3 --- Methylation-specific PCR (MSP) --- p.33 / Chapter 3.3.4 --- Bisulfite Genomic Sequencing (BGS) --- p.34 / Chapter 3.4 --- Construction of expression plasmids --- p.36 / Chapter 3.4.1 --- Construction of CDH11 expression vector --- p.36 / Chapter 3.4.2 --- Construction of PCDH10 expression plasmid --- p.38 / Chapter 3.4.3 --- Plasmid extraction --- p.39 / Chapter 3.5 --- Plasmid transfection --- p.41 / Chapter 3.6 --- Subcellular localization --- p.42 / Chapter 3.7 --- Function analyses --- p.43 / Chapter 3.7.1 --- Colony formation assay --- p.43 / Chapter 3.7.2 --- Wound healing assay --- p.44 / Chapter 3.8 --- Mechanism exploration --- p.44 / Chapter 3.8.1 --- Protein extraction and western-blot --- p.44 / Chapter 3.8.2 --- Dual-luciferase reporter assay --- p.47 / Chapter 3.9 --- Statistical analysis --- p.48 / Chapter Chapter 4 --- CDH11 functions as a tumor suppressor via modulating Wnt/β-catenin signaling and is frequently downregulated by promoter methylation --- p.49 / Chapter 4.1 --- The CpG island of CDH11 gene promoter --- p.50 / Chapter 4.2 --- CDH11 expression profile in normal tissues --- p.50 / Chapter 4.3 --- Frequent silencing of CDH11 by promoter methylation in multiple tumors --- p.51 / Chapter 4.4 --- Restoration of CDH11 expression by pharmacologic demethylation --- p.53 / Chapter 4.5 --- Frequent CDH11 methylation in primary tumors --- p.54 / Chapter 4.6 --- Function studies --- p.56 / Chapter 4.6.1 --- Ectopic expression of CDH11 inhibited tumor cell clonogenecity --- p.57 / Chapter 4.6.2 --- CDH11 induced tumor cell apoptosis --- p.57 / Chapter 4.6.3 --- CDH11 inhibited tumor cell migration --- p.58 / Chapter 4.7 --- CDH11 antagonized Wnt/β-catenin signaling --- p.59 / Chapter 4.8 --- Discussion --- p.60 / Chapter Chapter 5 --- Epigenetic inactivated tumor suppressor PCDH10 exerts tumor suppressive functions through modulating Wnt/β-catenin signaling and cell stemness in colon cancer --- p.66 / Chapter 5.1 --- PCDH10 was broadly expressed in normal tissues and frequently silenced in CRC cell lines --- p.67 / Chapter 5.2 --- Promoter methylation mediated PCDH10 silencing in CRC cell lines --- p.68 / Chapter 5.3 --- Frequent PCDH10 methylation in CRC primary tumors --- p.69 / Chapter 5.4 --- PCDH10 was located in cytoplasm and membrane --- p.70 / Chapter 5.5 --- Function Studies --- p.71 / Chapter 5.5.1 --- PCDH10 inhibited clonogenicity of tumor cells --- p.71 / Chapter 5.5.2 --- PCDH10 suppressed tumor cell mobility --- p.72 / Chapter 5.6 --- Mechanism exploration of PCDH10 in CRC --- p.72 / Chapter 5.6.1 --- PCDH10 antagonized Wnt/β-catenin signaling --- p.72 / Chapter 5.6.2 --- PCDH10 negatively regulated EMT and stemness of tumor cells --- p.74 / Chapter 5.6.3 --- PCDH10 inhibited RhoA signaling --- p.75 / Chapter 5.7 --- Discussion --- p.75 / Chapter Chapter 6 --- Conclusions and Future studies --- p.80 / Chapter 6.1 --- Conclusions --- p.80 / Chapter 6.2 --- Future studies --- p.82 / Reference List --- p.84

A two by six factorial design was used to investigate the effect of zinc and all-trans retinoic acid (RA) on the growth of two human breast cancer cell lines differing in their expression of metallothionein (MT) and of estrogen receptors; MCF7 cells express estrogen receptors, BT-20 cells do not. Cells were treated with zinc to induce MT then treated with six concentrations of RA. Cell proliferation, lipid peroxidation, MT protein, MT mRNA and glutathione concentrations were measured. / BT-20 cells expressed higher constitutive MT concentrations than MCF7 cells. MT was significantly induced by zinc treatment in BT-20 cells but not in MCF7 cells. Low RA concentrations stimulated growth proliferation but higher concentrations inhibited cell proliferation. High RA concentrations increased lipid peroxidation. There was a significant negative correlation between lipid peroxidation and cell proliferation. Growth inhibition and lipid peroxidation were reduced by zinc in BT-20 cells but not in MCF7 cells. Glutathione did not appear to be a significant factor. / Induction of metallothionein by zinc may modulate the growth inhibitory effects of all-trans retinoic acid in human breast cancer cells. One mechanism of growth inhibition may be through increased lipid peroxidation.

The signal transducer and activator of transcription (STAT) and protein inhibitor of STAT(PIAS) system represent an elegant regulatory mechanism of transcriptional control IN mammalian cytokine signalling. Abnormal activation of the system is associated with immune disorders and a large group of diverse tumours. PIAS3 is a multiple domain protein with distinct functions involved in regulation of cytokine-mediated gene activation pathways.Its over-expression significantly inhibits cell growth and renders cancer cells more sensitive to drugs. The objective of this study was to structurally and biochemically characterise the function of the PIAS3 protein using in silico, in vivo and in vitro analysis approaches.The conservation pattern of the PIAS protein family and critical conserved residues in the PINIT (Proline, Isoleucine, Asparagine, Isoleucine, Tyrosine) domain were identified. The PINIT domain model was generated based on the PINIT domain structure of yeast PIAS3 homologue Siz1 and structural determinants in the PIAS3-STAT3 interaction were evaluated.Guided by the in silico findings, in vivo analysis of the localisation of the PIAS3, mutantderivatives of PIAS3 (PIAS3-L97A, PIAS3-R99N, PIAS3-R99Q), PINIT and acidic domain was conducted. PIAS3 was completely localised in the nucleus while PIAS3 mutants appeared to exhibit diffuse cytoplasmic distribution. The PINIT domain was predominantly localised in the nucleus with some apparent perinuclear staining while the acidic domain exhibited a predominantly perinuclear staining pattern. Further analysis of the PINIT domain and the effect of the mutants on PIAS3-STAT3 interaction were assessed by in vitro analysis. Guided by in silico analysis, the PINIT domain and mutant derivatives of PINIT domain (PINIT-L97A, PINIT-R99N, and PINIT-R99Q) were heterologously expressed in Escherichia coli and subsequently purified using a combination of immobilized metal affinity and size exclusion based chromatography. The size and structural elements of the PINIT domain and its mutants were characterised. The 23 kDa PINIT domain was found to exist as a monomer in solution and its secondary structure was shown to consist of 66 % β-sheets by fourier transformed infrared spectroscopy consistent with the generated homology model.Using surface plasmonresonance spectroscopy (SPR) the PINIT domain was shown to bind to STAT3 in a specific concentration dependent manner. Recombinant PINIT-L97A,PINITR99N and PINIT-R99Q mutants, which exhibited similar structural integrity to the wildtype, were found to abrogate binding to STAT3. These findings suggest that these residues form part of a potential binding surface for stat3. In conclusion, this study has provided evidence that the PINIT domain is an important determinant of PIAS3 interaction with STAT3 and that the interaction is mediated by defined conserved residues directly involved in the PINITSTAT3 interaction.

Transcription factors (TFs) are key regulators of gene expression whose failure has been implicated in many diseases, including cancer. They bind at various sites at different specificity depending on the prevailing cellular conditions, disease, development stage or environmental conditions of the cell. TF binding specificity is how well a TF distinguishes functional sites from potential non-functional sites to form a useful regulatory network. Owing to its role in diseases, various techniques have been used to determine TF binding specificity in vitro and in vivo, including chromatin immuno-precipitation followed by massively parallel sequencing (ChIP-seq). ChIP-seq is an in vivo technique that considers how the chromatin landscape affects TF binding. Motif enrichment analysis (MEA) tools are used to identify motifs that are over-represented in ChIP-seq peak regions. One such tool, CentriMo, finds over-represented motifs at the center since peak calling software are biased to declaring binding regions centered at the TF binding site. In this study, we investigate the use of CentriMo and other MEA tools to determine the difference in motif enrichment attributed presence of Chronic Myeloid leukemia (CML)), treatment with Interferon (IFN) and Dexamethasone (DEX) compared to control based on Fisher’s exact test; using uniform peaks ChIP-seq data generated by the ENCODE consortium. CentriMo proved to be capable. We observed differential motif enrichment of TFs with tumor promoter activity: YY1, CEBPA, Egr1, Cmyc family, Gata1 and JunD in K562 while Stat1, Irf1, and Runx1 in Gm12878. Enrichment of CTCF in Gm12878 with YY1 as the immuno-precipitated (ChIP-ed) factor and the presence of significant spacing (SpaMo analysis) of CTCF and YY1 in Gm12878 but not in K562 could show that CTCF, as a repressor, helps in maintaining the required YY1 level in a normal cell line. IFN might reduce Cmyc and the Jun family of TFs binding via the repressive action of CTCF and E2f2. We also show that the concentration of DEX treatment affects motif enrichment with 50nm being an optimum concentration for Gr binding by maintaining open chromatin via AP1 TF. This study has demonstrated the usefulness of CentriMo for TF binding specificity analysis.

The termina1 enzyme in the linoleoyl-CoA desaturase enzyme complex, delta-6-desaturase was implied in the control of cell proliferation in cancer cells. One of the aims of this study was to isolate the terminal enzyme. It was decided that in order to isolate this enzyme it was first necessary to isolate the entire complex and then to enzymatically solubilise the first two components of the complex i e cytochrome b5 reductase and cytochrome b5 from the complex resulting in a pure delta-6-desaturase . The first two components were isolated and purified using simplified and easily reproducible methodologies which could be utilised in the final purification of delta-6- desaturase. The entire enzyme complex, linoleoyl-CoA desaturase was also isolated in a pure form and this pure complex was used to attempt to isolate delta-6-desaturase. The terminal enzyme was isolated with some cytochrome b5 still bound to it. The methods used had proven to be successful and with some modifications should yield a pure enzyme. Zinc and GLA were known to play a role in the inhibition of cancer cell proliferation and zinc was hypothesised to inhibit cell growth by stimulating the activity of the linoleoyl-CoA desaturase enzyme complex which is involved in the regulation of cell proliferation. GLA is the product of the reaction that this enzyme complex catalyses and GLA has been shown to inhibit cancer ce ll growth. The effect of GLA on cell growth and linoleoyl-CoA desaturase activity was thus investigated. Results showed that both zinc and GLA inhibited cell growth and that the combined addition of zinc and GLA generally resulted in the inhibition of cell growth and the activation of linoleoyl-CoA desaturase activity in the BL-6 cells while having a less pronounced effect on the LLCMK cells. The results of this study support the hypothesis that zinc may be a cofactor of linoleoyl-CoA desaturase.

No description available.

The concentration of insulin-like growth factor I (IGF-I) in tissue taken from human non-small cell lung carcinoma (NSCLC) is 1.4- to 7-fold higher than the concentration of IGF-I in the surrounding normal lung tissue and therefore IGF-I may be involved in the growth of NSCLC. In this study it was determined that NSCLC cell lines (A549, A427, SK-LU-1) expressed the type I IGF-I receptor protein and IGF-I stimulated the proliferation of low density plated (2000 cells/cm² growth area) carcinoma cells by 1.6- to 3- fold above control after a four day inCUbation period under serum-free conditions (A549, A427) or in the presence of 0.25% serum (SK-LU-1). In addition, when added to detergent-solubilized type I IGF receptors from A549 cells, IGF-I stimulated [1] a dose-dependent increase in the autophosphorylation of the type I IGF receptor, and [2] a dose-dependent increase (1.5- to 4-fold) in the phosphorylation of a tyrosine kinase-specific substrate. These results suggest that the growth promoting activity of IGF-I for the lung carcinoma cells was mediated through the activation of the type I IGF receptor. / Ph. D.

published_or_final_version / Pathology / Doctoral / Doctor of Philosophy