Proteomic Identification and Characterization of Proteins That Are Associated with Malignancy of Esophageal Cancer Cells

This dissertation, "Proteomic Identification and Characterization of Proteins That Are Associated With Malignancy of Esophageal Cancer Cells" by Zhen, Cai, 蔡貞, was obtained from The University of Hong Kong (Pokfulam, Hong Kong) and is being sold pursuant to Creative Commons: Attribution 3.0 Hong Kong License. The content of this dissertation has not been altered in any way. We have altered the formatting in order to facilitate the ease of printing and reading of the dissertation. All rights not granted by the above license are retained by the author. Abstract: Abstract of thesis entitled PROTEOMIC IDENTIFICATION AND CHARACTERIZATION OF PROTEINS THAT ARE ASSOCIATED WITH MALIGNANCY OF ESOPHAGEAL CANCER CELLS Submitted by CAI Zhen for the degree of Doctor of Philosophy at The University of Hong Kong in October 2007 Esophageal squamous cell carcinoma (ESCC), a predominant histological type of esophageal neoplasma, is characterized by poor prognosis and rapid clinical progression, with a high frequency of metastasis and recurrence. Ambiguity in molecular mechanisms of etiopathologenesis and disease progression has made it difficult to identify markers for early diagnosis and specialized drug targets. In this study, a combination of 2DE-based comparative proteomics and biochemical methods was employed to analyze the proteome of different experimental models representing tumorigenic and metastatic processes of ESCC, in an attempt to elucidate malignant progression globally and to identify potential protein molecules that might serve as early diagnostic markers, prognosis indicators or treatment targets. Differentially expressed proteins between immortalized esophageal epithelial NE1 cells and cancerous HKESC-2 cells or between xenografts derived respectively from primary ESCCs and metastatic lymph nodes are thought to be associated with malignant phenotype or special to the metastatic characteristics of ESCC. Much attention had been paid to cytoskeletal and associated proteins, due to their ability to govern cell motility and act as signal integrated platform, characteristics especially important in malignant transformation and progression. Several clusters of altered proteins were identified and the expression changes of interesting proteins were further validated in a panel of esophageal origin cell lines and clinical specimens including paired tumor/non-tumor tissue and concomitant primary ESCCs and metastases. CK8 and CK18 were significantly up-regulated in esophageal malignancies, concomitant with changed expression profiles. With metastasis models, CK8 was further found with an obvious increase in expression with similar pattern changes in metastatic tumor-origin xenografts as compared to their primary ESCC derived counterparts, suggesting that CK8 plays a very important role, through either the expression level or post-translational modifications, in ESCC progression. Elevated expressions of galectin-7 and S100A9 in metastatic disease were also confirmed by Western blotting and IHC. Results of IHC further indicated that the translocation of these two proteins from cytoplasma to the nucleus might have important impacts on metastasis of esophageal malignancies. Maspin is one of the identified proteins found to be down-regulated in both tumorigenic and metastatic models. To test its putative effects in ESCC metastasis process, an EMT model was established with maspin-null esophageal cancerous cell line EC109 to mimic in vitro some aspects of the metastasis process. It was found that the stable introduction of maspin into EC109 cells was able to antagonize EGF-induced EMT in EC109 cells, leading to a decreased ability in colony formation. Results from proteomic analysis showed that a group of glycolytic enzymes were decreased in maspin- transfected cells, indicating lower glycolysis of these cells. We therefore propose that the metastasis-suppressive effect of maspin may be a consequence of the re