Nasopharyngeal carcinoma (NPC), endemic in southern China and Southeast Asia, was ranked 7th as the most common new malignancy in Hong Kong. Metastatic and recurrent NPC have a poor prognosis despite recent advancement in medicine. Inactivation of tumor suppressor genes (TSGs) through the loss of chromosomal regions is frequently reported in NPC. With the recent discovery of cancer stem cells (CSCs), which are refractory to current therapies, a new paradigm shift in the perspective of cancer therapy development has emerged. For the first time, this study aims to unravel the complexity of NPC tumorigenicity for identifying more effective targets by studying the possible interplay between CSCs and TSGs. NPC cell lines had different expression profiles of CSC markers, confirming not all CSC markers are applicable to every tumor type. Although CD24/CD44 were expressed in NPC, however CD24+CD44+ NPC cells did not initiate tumor formation. By utilizing a cancer hybrid cell model with a transferred single copy of chromosome 3, physiological β-catenin up-regulated core stem cell markers through the activation of Wnt signaling pathway in NPC. Moreover, the down-regulation of β-catenin suppressed chemoresistance and inhibited cell proliferation, colony formation, angiogenesis, the epithelial-mesenchymal transition (EMT) process, and the tumor microenvironment factors. Amongst the tumor microenvironment factors, chemokine Rantes and matrix metalloproteinase were down-regulated when β-catenin was knocked down. Therefore, activation of Wnt signaling provide an alternative platform for identifying putative CSCs in NPC, leading to the identification of several prospective CSC markers in NPC. Down-regulation of IKBB, a NF-KB inhibitor, in the majority of NPC patients indicated that IKBB plays a prominent role as a TSG in NPC. In this study, IKBB was found to exert its tumor suppressive functions by abrogating tumor formation, cell migration, invasion and angiogenesis. Angiogenic factors, including Rantes, Upar, IL6 and IL8, were significantly down-regulated by IKBB. In addition, IKBB also suppressed the binding activity of NF-KB. The involvement of Akt/Gsk-3β pathways was also observed. Taken together, IKBB regulated NPC tumorigenesis through NF-KB/Akt/Gsk-3β and interaction with tumor microenvironment. Collectively, this study demonstrated that the progression of NPC is not simply initiated by a single signaling pathway, but a dynamic and complex interplay between multiple signaling networks and the tumor microenvironment. NPC tumorigenesis is hypothesized to be driven by orchestrated interaction between CSCs and TSGs through crosstalk with the tumor microenvironment. Amongst the major players in the tumor microenvironment, Rantes/CCL5, IL6, and the matrix metalloproteinase are envisaged to induce angiogenesis, EMT, and metastasis in NPC. This dynamic intercommunication between CSCs and tumor suppressor IKBB signaling networks may shed better insights on modulation of the major hallmarks of cancer in orchestrating NPC development. The modulation of the major hallmarks of cancer by CSCs and IKBB, a TSG, involves promotion of aberrant proliferation, enhancement of invasion and metastasis, induction of angiogenesis, circumvention of tumor suppressors, and prevention of cell death. Taken together, selective and synergistic co-targeting these signaling networks and the tumor microenvironment will provide a more effective new modality of treatments for NPC. / published_or_final_version / Clinical Oncology / Doctoral / Doctor of Philosophy

Neuroblastoma is a childhood solid tumor of a unique propensity to either regress spontaneously or grow relentlessly. Emerging evidence indicated that neuroblastoma contains heterogeneous populations of cells, and commitment of these cells to neuronal lineage may result in aggressive progression in patients, whereas to fibromuscular lineage may give a favorable outcome. However, mechanism(s) controlling the lineage commitment of neuroblastoma cells remains to be identified. Our preliminary data suggested that Kr?ppel-Like Factor 4 (KLF4) might promote neuroblastoma regression. KLF4 is a transcription factor regulating a variety of cellular functions, including proliferation and cell cycle progression. Recent studies have demonstrated that KLF4 may act as both tumor suppressor and oncogene in a cell-context dependent manner. Importantly, our preliminary data showed that low KLF4 expression is highly associated with poor clinical outcomes of the neuroblastoma patients. In addition, we found that overexpression of KLF4 suppresses neuroblastoma cell growth accompanied with loss of tumorigenicity. Morphologically, KLF4 overexpressing cells changed their morphologies to become epithelial-like, strongly substrate-adherent and expressing smooth muscle marker. Therefore, we hypothesized that KLF4 exerts its effects through two ways, it may (i) function to inhibit cell growth and reduce tumorigenicity; and (ii) promote differentiation of the neuroblastoma cells to the non-tumorigenic, fibromuscular-like cells. RT-PCR data revealed the differential expression of KLF4 in 11 neuroblastoma cell lines. In particular, a modest expression was found in Be(2)C, a cell line which was formerly demonstrated to differentiate and form tumor in mice xenograft assay. It was therefore chosen as the study model. To assess the effects of KLF4 knockdown on tumor growth, stable knockdown clones from Be(2)C cells were established by lentiviral transduction of KLF4-targeting shRNA. In parallel, clones that stably expressed non-target shRNA were used as controls. After the transduction, two stable knockdown clones showing significant KLF4 downregulation were isolated from single colony (monoclonal stable clones) and a pool of cells (polyclonal stable clones) respectively. The cell proliferation and growth rate of the stable clones were then measured by 5-bromo-2’-deoxyuridine (BrdU) proliferation assay and growth curve assay. The results have indicated that both monoclonal and polyclonal stable KLF4 knockdown clones grow faster than the control clones. In order to examine the tumorigenicity in vivo, the stable clones were xenotransplanted to severe combined immunodeficient mice. The stable KLF4 knockdown clones showed a significant higher growth rate and formed a larger tumor. The stable clones were also treated with BrdU for four weeks for differentiation towards fibromuscular lineage. As anticipated, the control clones showed fibromuscular features, like more flattened and epithelial-like morphology. In contrast, the stable KLF4 knockdown clones failed to present the fibromuscular features after treatment. In addition, immunocytochemistry staining of SMA and quantitative analysis of the immunocytochemistry further confirmed that only the control clones showed higher SMA expression after BrdU treatment, while there is no change in the SMA expression in the stable KLF4 knockdown clones. These results demonstrated that KLF4 functioned by inhibiting neuroblastoma cell proliferation and growth, reducing the tumorigenicity, and it was required for fibromuscular differentiation. / published_or_final_version / Surgery / Master / Master of Philosophy

Esophageal squamous cell carcinoma (ESCC), the major histological subtype of esophageal cancer, is one of the most common malignancies with poor prognosis in the world. Despite continued development of diagnosis and treatment, ESCC remains the sixth leading cause of cancer death worldwide. Current treatment regimens in ESCC are often characterized by ineffectiveness and poor selectivity. Therapeutic methods directed at cancer-associated genes or cancer stem cells (CSCs) may be effective approaches to cure this deadly cancer. Therefore, this study aims to identify specific ESCC-related genes and cancer stemness genes which help us to develop new targeted agents to achieving objective, long-lasting therapeutic responses in ESCC. To obtain an accurate overview of genetic changes occurring in ESCC patients, our group performed microarray-based mRNA expression profiling and high-throughout transcriptome sequencing (RNA-Seq) to compare differentially expressed genes between ESCC tumors and their corresponding non-tumorous tissues. Prostate stem cell antigen (PSCA) was considered to be a candidate of primary interest due to significantly reduced expression in both microarray and RNA-Seq data. In this study, we examined the role of PSCA on the pathogenesis of esophageal cancer. Our results showed that PSCA was frequently down-regulated in ESCC. Its expression was negatively regulated by transcription factor SOX5. Also, we provided evidence that down-regulation of PSCA was associated with poor clinical outcomes of patients with ESCC. Both in vitro and in vivo assays revealed that PSCA could arrest cell cycle progression and promote differentiation. To further elucidate the mechanism involved in biological function of PSCA, we performed co-immunoprecipitation and mass spectroscopy to identify proteins that associate with PSCA. This study found that RB1CC1, a key signaling node to regulate cellular proliferation and differentiation, interacted specifically with PSCA both in vitro and in vivo. Binding of PSCA and RB1CC1 in cytoplasm resulted in stabilization and translocation of RB1CC1 into nucleus and then further regulates the crucial cell cycle and differentiation genes. Furthermore, in order to identify the cancer stemness genes specifically expressed in CSCs of ESCC, we utilized gene expression analysis to profile 34 stemness-associated genes in ESCC specimens. Developmental pluripotency associated 4 (DPPA4), a well known pluripotent marker of stem cell, was considered as the best candidate. Our following histopathological study demonstrated that DPPA4 rigorously marked the rare CSCs, in contrast to core stemness factors (OCT4 and SOX2) and previous reported CSC markers (CD90 and CD44), which expressed in a large population of cancer cells. Moreover, the expression of DPPA4 was also found to have prognostic value in ESCC, as the appearance of DPPA4+ cells was significantly associated with poor differentiation, advanced stage and higher incidences of lymph node metastasis. Finally, our functional studies showed that ESCC cells expressing exogenous DPPA4 conferred an enhanced ability to initiate tumor, self-renew, resist chemotherapy and metastasize through lymphatic system. In summary, this study provide evidence indicating that novel tumor suppressor gene PSCA and cancer stemness gene DPPA4 may contribute to the development and progression of ESCC. Additionally, they may serve as potential targets for development of effective therapeutic strategies. / published_or_final_version / Clinical Oncology / Doctoral / Doctor of Philosophy

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published_or_final_version / Medical Sciences / Master / Master of Medical Sciences

The human wildtype p53 tumor suppressor gene contains a polymorphism at amino acid residue 72 which results in either an arginine (p53 Arg-72) or proline (p53 Pro-72) at this codon. In the present study I have examined this polymorphism at the molecular level to determine whether differences exist in the biochemical functions of these two p53 variants. No differences were observed in their sequence-specific DNA binding abilities, nor in their ability to be targeted by HPV-18 E6 oncoprotein for degradation by ubiquitination in vitro. However, differences were observed in the ability of these two variants to function as transcriptional activators: p53 Pro-72 was more transcriptionally active than p53 Arg-72. I propose that the polymorphism at codon 72 may affect the structure of the N-terminal transactivation domain of the p53 protein, which would then have an effect on the ability of these variants to interact with transcription factors in order to initiate transcription of target genes and function as a tumor suppressor.

Thesis (Ph. D.)--University of Hong Kong, 2008. / Also available online.

Thesis (Ph. D.)--University of Hong Kong, 2008.

Thesis (Master)--İzmir Institute of Technology, İzmir, 2005. / Keywords: Antioxidants, gene expression, tumor suppressor genes. Includes bibliographical references (leaves. 51-63).

Thesis (Ph.D.)--Georgetown University, 2008. / Includes bibliographical references.