Use this URL to cite or link to this record in EThOS: http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.616755
Title: Mechanisms of colorectal cancer pathogenesis : role of hypoxia
Author: Thairu, Ngayu Munga
Awarding Body: Imperial College London
Current Institution: Imperial College London
Date of Award: 2013
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Abstract:
Colorectal cancer (CRC) is the third most common cancer worldwide Hypoxia plays a pivotal role in cancer, regulating cellular processes such as angiogenesis via the Hypoxia Inducible Factor (HIF) pathway. HIF-1α and HIF-2α, isoforms of the α-subunit, were previously thought to be functionally redundant, but mounting evidence supports their divergent roles in many cancers. In CRC their relative roles remain unclear. This study aimed to elucidate their relative contribution to hypoxic regulation of CRC using the Caco-2 cell-line. Ex vivo cultures of primary cells isolated from CRC tissue were used to validate the Caco-2 data. Caco-2 cells were stimulated with hypoxia (1% O2) or the hypoxia-mimetic dimethyloxaloylglycine (DMOG), with normoxia (21% O2) controls. Expression of known hypoxia-induced genes was quantified by polymerase chain reaction (PCR) and a PCR-based array was used to further characterise angiogenic genes. Protein expression was determined using Western Blotting and ELISA. The effect of selective HIF-isoform knockdown on gene expression was evaluated. Tumour-derived cultures (TDCs) were established using tissue obtained from surgically resected CRC specimens. mRNA expression of epithelial cell markers (Ep-CAM, VE-Cadherin) was quantified by Q-PCR, and protein expression of the CRC tumour marker carcinoembryonic antigen (CEA) measured by ELISA. TDCs were exposed to hypoxia, and gene expression relative to normoxia was quantified by Q-PCR and PCR-based array. In Caco-2 cells, hypoxia upregulated both HIF isoform proteins, inducing genes involved in angiogenesis (VEGF, ANGPTL-4, EFNA-3, TGF-β1), metabolism (CA-IX, GLUT-1) and apoptosis (BNIP-3), with mRNA changes reflected at protein level. Three novel hypoxia-induced angiogenesis genes (ANGPTL-4, EFNA-3, TGF-β1) were identified. Hypoxia-induced ANGPTL-4, BNIP-3 and TGF-β1 expression was reduced by siHIF-1α only, while EFNA-3 and VEGF expression was reduced by both siHIF-1α and siHIF-2α. TDCs expressed epithelial CRC cell markers, and showed similar hypoxia-induced angiogenesis gene expression to Caco-2 cells, although there was significant inter-donor variability.
Supervisor: Paleolog, Ewa ; Kiriakidis, Serafim ; Dawson, Peter Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID: uk.bl.ethos.616755  DOI: Not available
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