Functional analysis of N-MYC downstream regulated gene 1 (NDRG1) in Oesophageal squamous cell carcinoma

Doctoral Thesis

2009

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University of Cape Town

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Abstract
Oesophageal squamous cell carcinoma (OSCC) ranks as one of the deadliest tumours with a high incidence in developing countries in the areas of Southern Africa, Middle East and Far East. Moreover, its unfavourable prognosis is further complicated by the lack of knowledge about the molecular biology of this disease. In this thesis, we describe our work analysing the function of N-myc downstream regulated gene 1 (NDRG1, also known as Cap43 or Drg-1) in the neoplastic progression and maintenance of OSCC. Although NDRG1 has previously been implicated in breast, prostate, colon and liver carcinoma, the exact role of NDRG1 in OSCC still remains unclear. According to the immunohistochemical analysis of clinical OSCC tissue samples (n=52), NDRG1 expression was gradually increased in tumour tissue versus normal, indicating the potential involvement of NDRG1 in the neoplastic progression of OSCC. We next performed ectopic NDRG1 gain-of-function and loss-of-function studies using transfectants established from transduced OSCC cell lines (KYSE30 and KYSE150) by lentiviral vector mediated gene delivery. In KYSE30 cells, although no substantial effects on in vitro cell proliferation and differentiation were observed with altered NDRG1 expression, the ectopic overexpression of NDRG1 was found to be positively linked to metastasis, angiogenesis and apoptotic evasion as measured in cell culture. Accordingly, in the nude mouse xenograft model system, NDRG1 overexpression promoted the in vivo growth and metastasis of KYSE30 derived xenografts, which could be attributed to the reduced apoptotic and enhanced angiogenic activities promoted by this gene. Nevertheless, no significant phenotypic changes were observed in response to NDRG1 knock-down, suggesting that this gene was not essential for the neoplastic progression of OSCC. Moreover, null effect of either ectopic NDRG1 overexpression or knock-down were observed in KYSE150 cells, indicating ix that the function of NDRG1 may be largely dependent on the cellular context (Chapter 2). In addition to direct functional assays, evidence from analysing the regulation pattern of NDRG1 in OSCC cells was also presented to provide clues to indirectly predict the function of NDRG1 in OSCC. In Chapter 3, we demonstrated that NDRG1 could be actively regulated by various oncogenic stimuli such as cellular stress (genotoxicity and hypoxia) and mitogenic factors (EGF and IGF). Although these oncogenic regulatory effects on NDRG1 expression in OSCC cells may be dichotomous, the functional significance of NDRG1 upregulation, especially by hypoxia and EGF signalling, is highlighted. In our studies, the regulatory pattern of NDRG1 in OSCC is highly consistent with its oncogenic function revealed in ectopic studies (Chapter 2), further suggesting that phenotypic changes observed in the functional studies may not be artifactual, but may reflect the role of NDRG1 in the neoplastic progression of OSCC in physiological conditions. Taken together, our current data implicate NDRG1 as an effective but non-essential promoter in the neoplastic progression of oesophageal squamous cell carcinoma. Although the mechanism still needed to be further explored, our study suggests important clues regarding these mechanistic roles considering the impact of this gene on apoptosis, metastasis and angiogenesis.
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