Song, Qingkun. / Thesis (Ph.D.)--Chinese University of Hong Kong, 2011. / Includes bibliographical references (leaves 144-157). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Abstract also in Chinese.

A thesis submitted to the Faculty of Science, University of the Witwatersrand, in fulfilment of the requirements for the degree of Doctor of Philosophy Johannesburg, 2011. / The phosphotidylinositide-3-kinase (PI3K) pro-survival signalling pathway is critical in the development of cancer. Major contributors to the proliferative and/or anti-proliferative signalling in human oesophageal squamous cell carcinoma (HOSCC) are currently unknown. Based on the Ser473 phosphorylation state of PKB (pPKB), this study dissects the overall activation status of the PI3K/PKB pathway. Despite the prevalent membrane expression of PI3K determined through western blotting and immunofluorescence, pPKB levels were shown to be surprisingly low. Activation of EGFR did not produce a hyperactivation of the PI3K/PKB pathway. Neither PI3K nor PKB sequence isolated from the 5 HOSCC cell lines possessed any of the ―hotspot‖ mutations described previously for other tumours. Inhibiting phosphatase protein 2A (PP2A), an integral antagonist of PKB, indicated that its activity in respect of PKB is diminished in HOSCC cells. Despite the low concentration of pPKB, the reciprocal relationship with PTEN expression was not evident in the WHCO and SNO HOSCC series. Moreover, reversible oxidization and inhibition of PTEN served to augment the activation of the PI3K/PKB pathway. Since oxidation of PTEN is imperative for effective signal propagation from activated EGFR and PI3K, these data reveal an aberrant EGFR-PI3K-H2O2 mediated PTEN inhibition in HOSCC. Allied to this discovery, was the finding that HOSCC cells are highly susceptible to oxidative stress induced by H2O2. This was suggested to play an essential part in maintaining the low PI3K/PKB activation status. Although the decrease in PTEN activity was required for the induction of pPKB, PTEN may not be the only limiting component for the activation of the PI3K/PKB pathway in HOSCC. In addition to its overexpressed EGFR status, the WHCO and SNO HOSCC series have the propensity to appropriate nuclear β-catenin. Interruption of the PI3K/PKB signalling pathway caused a small, yet significant, depression in the nuclear localization of β-catenin in 3 of the HOSCC cell lines. Together, this work greatly expands our understanding of the major influences behind the proliferative and/or anti-proliferative signalling in HOSCC, primarily that, the EGFR overexpression status does not propagate these transforming capabilities via activation of the PI3K/PKB pathway, and that this may be a reflection of its transformation potential. The findings derived from this study are likely to have a profound impact on future therapeutic targets for this disease.

published_or_final_version / Medical Sciences / Master / Master of Medical Sciences

published_or_final_version / Surgery / Master / Master of Research in Medicine

published_or_final_version / Clinical Oncology / Master / Master of Philosophy

published_or_final_version / Clinical Oncology / Doctoral / Doctor of Philosophy

Background Esophageal carcinoma mainly affects middle aged to elderly males. It ranks the ninth most common cancer world-wide. The main histological types are squamous cell carcinoma and adenocarcinoma. In Hong Kong, esophagus squamous cell carcinoma (ESCC) is by far the more common. BARX2 is a human homeobox gene located at 11q24-q25, encoding a protein of 254 amino acids. Recent researches show that its expression in breast cancer promotes cellular invasion. Objectives The study aimed to test the hypothesis that BARX2 is a prognostic marker in ESCC. BARX2 expression in ESCC was correlated with patient survival and other clinicopathologic parameters in a cohort of patients. Material and Methods Records of ESCC patients were obtained retrospectively from the computerized database of Queen Mary Hospital. ESCC patients, who underwent esophagectomy in the hospital from 1998 to 2005 but without receiving prior chemotherapy or radiotherapy directed to the tumor, were selected. Tumor staging was done according to the 6th edition of AJCC Cancer Staging Manual. Immunohistochemical staining for BARX2 expression was performed on paraffin sections of the primary ESCC tissues sampled in a tissue microarray constructed for research purposes. The pattern of BARX2 expression in nucleus and cell cytoplasm of tumor cells was recorded and the staining intensity scored on a 4-point scale. The scores were statistically analyzed together with the various clinicopathologic parameters. BARX2 expression and patient survival time were analyzed by the log-rank test. Results A total of 78 ESCC patients were recruited. At the time of data analysis, 52 (66.7%) patients were dead. The overall median survival of patients was 14.3 months. BARX2 was found to be mainly expressed in the cytoplasm of tumor cells while non-tumor epithelium showed strong nuclear expression. Patients with high level BARX2 expression had short survival time, though the difference did not reach statistical significance (p=0.075). Within the subgroup of lower T-stage ESCC (T1-3), high level BARX2 expression was significantly associated with shorter survival time (p=0.042). However, differential BARX2 expression did not affect survival time within the group of patients who had advanced stage (T4) disease (p=0.525). In patients who had no regional lymph node metastasis (N0), high level BARX2 expression was associated with shorter survival time (p=0.023). However, when patients had regional lymph node metastases (N1), BARX2 expression did not affect patient survival time (p=0.533). Patients whose ESCC showed moderate differentiation in a three-tier tumor grading system, when accompanied with low level BARX2 expression, had longer survival time (p=0.029). However, BARX2 expression did not affect survival time when ESCC showed either well differentiation (p=0.462) or poor differentiation (p=0.637). Multivariate analysis showed patient age and T-stage to be the only two independent parameters of prognostic significance (p=0.025 and p=0.036 respectively). Conclusions BARX2 expression in ESCC was aberrant and mainly cytoplasmic. It was inversely correlated with patient survival time in early ESCC disease (T1-T3 or N0). BARX2 expression evaluated by immunohistochemistry could be a useful and practical prognostic marker of ESCC in its early stages, when the proper decision on treatment would be critical for the patients. / published_or_final_version / Pathology / Master / Master of Medical Sciences

Esophageal cancer is the third most common digestive tract malignancy. Along with surgery, genotoxic drugs (e.g. cisplatin) and radiotherapy are the mainstays of treatment for this disease. Environmental factors and environmental stress-induced responses contribute to esophageal tumorigenesis and chemoresistance. Studying key molecules in stress-induced signal pathway can help unravel the underlying mechanisms and discover rational therapeutic targets. Cyclin D1 is DNA damage response protein. Genotoxic stress induces rapid cyclin D1 degradation and the molecules mediating this response are cell-type dependent. The first part of this study investigated the changes of cyclin D1 expression in response to genotoxic stress in immortalized esophageal epithelial cells, which are experimental models commonly used to study the early events of cancer development. The results showed that cyclin D1 underwent rapid proteasomal degradation before p53-induced p21 accumulation, which substantiates that cyclin D1 plays a role in eliciting cell cycle arrest very early in the DNA damage response. FBXO31 and FBX4, two F-box proteins previously reported to mediate cyclin D1 degradation, were found to be accumulated and unchanged, respectively, after ionizing irradiation in immortalized esophageal epithelial cells and esophageal squamous cell carcinoma (ESCC) cell lines. Yet, knockdown of FBXO31 did not rescue rapid cyclin D1 degradation upon UV or ionizing irradiation. This led to the hypothesis that accumulation of FBXO31 may have novel functions beyond mediating cyclin D1 degradation in cells responding to genotoxic stress. The second part of this study explored the function of FBXO31 in genotoxic stress response. The accumulation of FBXO31 in cancer cells after exposure to various genotoxic stresses was found to coincide with p38 deactivation, giving the clue that FBXO31 may negatively regulate this important pathway. Further studies revealed that knockdown of FBXO31 resulted in sustained activation of stress-activated MAPKs (SAPKs) p38 and JNK, as well as increase in UV-induced cell apoptosis, whereas overexpression of FBXO31 had opposite effects. The inhibitory role of FBXO31 on SAPK activation and apoptosis was confirmed by shRNA rescue experiments. Consistent with the observed anti-apoptotic effect, soft agar, colony formation and in vivo xenograft experiments showed that FBXO31 had oncogenic function in ESCC. Moreover, in vitro and in vivo results showed that knockdown of FBXO31 could sensitize ESCC cells to cisplatin treatment. The mechanism underlying the inhibition of SAPKs by FBXO31 was investigated in the third part of this study. Co-immunoprecipitation results showed that FBXO31 could interact with MKK6 (a p38 activator), but not p38, JNK1, or other MAP2Ks. FBXO31 was found to be co-localized with MKK6 in the cytoplasm. Mapping of interaction domains of FBXO31 revealed that aa 115-240 and aa 351-475 were responsible for binding to MKK6. Further study found that binding of FBXO31 to MKK6 could facilitate the K48-linked polyubiquitination and degradation of MKK6. Taken together, the results of this study showed that FBXO31 accumulation upon genotoxic stress can promote the degradation of MKK6 via K48-linked ubiquitination, thereby inhibiting SAPK activation and protecting cancer cells from genotoxic stress-induced apoptosis. FBXO31 may be a potentially useful therapeutic target to overcome chemoresistance in cancer therapy. / published_or_final_version / Anatomy / Doctoral / Doctor of Philosophy

published_or_final_version / Medical Sciences / Master / Master of Medical Sciences

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