published_or_final_version / Surgery / Master / Master of Surgery

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

published_or_final_version / abstract / toc / Surgery / Master / Master of Surgery

M.Sc.

M.Sc. / Statistics provided by the World Health Organization state that cancer accounted for 7.9 million deaths worldwide in 2007, with numbers expected to increase to over 12 million by the year 2030. The transformation of a normal cell to a malignant tumour is known to be the result of a set of several key mutations in the genome of a normal cell, resulting in several unique properties including the evasion of programmed cell death, or apoptosis. Exacerbation of this cell death evasion can occur by overexpression of cell survival effectors such as heat shock proteins (Hsps), which are a family of highly conserved proteins that are rapidly induced in response to a variety of stresses in order to protect the cell from death. These proteins perform this function both by assisting in protein folding and therefore acting as molecular chaperones and also by directly interacting with the apoptotic machinery to prevent the initiation of cellular death. Various Hsps interfere at a range of sites in the intrinsic apoptotic pathway, both upstream of the mitochondria, and downstream at the sites of caspase activation. Similarly, Hsps also interfere at various sites in the extrinsic pathway, the caspase-independent pathways, and also function to promote the activity of survival pathways. Nonsteroidal anti-inflammatory drugs (NSAIDs) are well known for their anti-inflammatory properties via inhibition of cyclooxygenase (COX) enzymes. These drugs have also been shown to induce apoptosis in a variety of cancer cell lines as well as decrease the risk of the development of various cancers. Interestingly, NSAIDs have additionally been shown to have the curious property of activating the heat shock transcription factor (HSF1) at concentrations much higher than that required for inhibition of COX activity. The combination of NSAIDs and hyperthermia has resulted in seemingly contradictory evidence, where some studies show that this combination leads to thermotolerance and resistance to further treatments, whilst other studies have shown that this combination directly leads to cell death or indirectly sensitizes cells to subsequent stress.

Metastasis accounts for more than 90% of cancer-related mortality, and thus, there is a compelling need for innovative therapeutic breakthroughs. TP53 mutations are detected in up to 80% of esophageal squamous cell carcinomas (ESCCs), the major subtype of esophageal cancer and one of the most lethal cancers worldwide, as well as in other SCCs. These mutations in turn correlate with poor patient prognosis and high metastatic rates. To elucidate novel mutant p53-dependent mechanisms in promoting ESCC metastasis, we generated a mouse model combining genetic and carcinogenic approaches: We treated the L2-Cre (esophageal specific promoter); LSL-Trp53R172H/-, Trp53-/- or Trp53+/+; Rosa26LSL-YFP mice with a carcinogen 4-NQO, and isolated primary and metastatic tumor cells that vary in their p53 statuses. We have shown that ESCC cells with Trp53R172H exhibit greater metastatic capabilities compared to the tumor cells harboring Trp53-/-, indicating gain-of-function (GOF) activity. Through comprehensive RNA-seq and cytokine array analyses, we identified that Colony-stimulating factor-1 (Csf-1) is significantly upregulated in a p53-R172H-dependent manner in metastatic lung lesions of ESCC. p53-R172H binds to the promoter region of Csf-1 locus in metastatic ESCC cells. Our findings demonstrate that p53-R172H-dependent Csf-1 signaling through its cognate receptor Csf-1r enhances tumor cell invasion and lung metastasis by utilizing complementary genetic and pharmacological approaches. This mechanism is mediated in part through Stat3 phosphorylation and epithelial-to-mesenchymal transition (EMT). These findings are further supported through in vivo targeting of Csf-1r. In addition, high levels of CSF-1 also correlate with mutant p53 in ESCC Tissue Microarrays (TMAs) and The Cancer Genome Atlas (TCGA) datasets. Our CUT&RUN-seq analysis on ESCC tumor cells revealed that both the Csf-1 locus and EMT-associated genes are enriched with histone 3 lysine 27 acetylation (H3K27ac). This enrichment creates a permissive environment for the interaction between Brd4 and p53-R172H, thereby regulating Csf-1 transcription. Notably, Brd4 interacts specifically with p53-R172H. Inhibiting Brd4 not only decreases tumor invasion and lung metastasis, but also reduces circulating Csf-1 levels in blood serum in vivo. Overall, our results establish a novel p53-R172H-dependent Brd4-Csf-1 signaling axis that facilitates lung metastasis in ESCC and underscores the GOF properties of p53-R172H. Our discoveries identify therapeutic vulnerabilities in metastatic ESCC, which can be applicable to other SCCs with similar transcriptomic and epigenetic profiles. These insights pave the way for developing therapeutic strategies for this difficult-to-treat disease.

Yu, Le. / Thesis (Ph.D.)--Chinese University of Hong Kong, 2009. / Includes bibliographical references (leaves 171-198). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Abstracts in English and Chinese.