Plasma membrane calcium ATPase during colon cancer cell differentiation and in colon cancer

Colon cancer is the third most common type of cancer, with high mortality throughout the world. During tumorigenesis, normal cells transform into tumour cells following changes in the expression of oncogenes and/or tumour suppressor genes, which are involved in many processes including the cell cycle, differentiation and apoptosis. An imbalance in the regulation of proliferation and differentiation in colon epithelial cells is usually associated with the development of colon cancers. Uncontrolled proliferation with a lack of differentiation is one of the major characteristic features of cancer cells and a remodelling of the Ca2+ signalling is linked to these pathways. Among the Ca2+ transporting proteins, P-type Ca2+-ATPases, the plasma membrane Ca2+ ATPase (PMCA) pump, has a high-affinity for Ca2+ and is involved in the efflux of Ca2+ against the electrochemical gradient from the cytosol across the extracellular space. Four PMCA isoforms have been identified. PMCA1 and 4 are expressed in most tissues. Changes in the expression of PMCA have been documented in breast cancer cells, whereas the expression profile of PMCA isoforms in colon cancer cells remains unknown. Up-regulation of another P-type Ca2+-ATPase, expressed in the endoplasmic reticulum, SERCA3, occurs during the differentiation of colon cancer cell lines and is down-regulated in colon cancers. Changes in PMCA expression have not been assessed during colon cancer cell differentiation. The first part of this thesis describes the analysis of the expression profile of PMCA during colon cancer cell differentiation. Both PMCA mRNA and protein levels were assessed in differentiated HT-29 cells by real time RT-PCR and western blotting analysis, respectively. The results showed changes in PMCA4 expression, whereas changes in the expression of PMCA1 were not associated with differentiation of HT-29 cells. PMCA mRNA levels were also reduced in some colon cancers suggesting a remodelling of PMCA-mediated Ca2+ efflux during colon carcinogenesis. The second part of this thesis involved exploring the functional role of PMCA4 in Ca2+-mediated signalling pathways such as differentiation, proliferation and apoptosis. PMCA4 expression was altered in HT-29 colon cancer cells via transient and stable over-expression of a PMCA4 expressing plasmid or siRNA-mediated silencing of PMCA4. An increase in the PMCA4 level did not alter or induce differentiation of HT-29 cells. Hence, up-regulation of PMCA4 expression may be a consequence rather than a cause of HT-29 colon cancer cell differentiation. PMCA4-mediated reduction in proliferation was observed in HT-29 colon cancer cells where PMCA4 was stably over-expressed. Stable PMCA4 over-expression was also associated with the down-regulation of the transcription of the early response gene, FOS. Despite the apparent augmentation of cytosolic Ca2+ responses to G-protein coupled receptor Ca2+ mobilizing agents, the sensitivity of cells to the apoptotic inducing agents such as TRAIL and/or CCCP was not affected following siRNA-mediated PMCA4 inhibition in HT-29 cells. Collectively this thesis describes PMCA isoform-specific changes during differentiation of HT-29 colon cancer cells and alterations in PMCA levels in some colon cancers.Evidence is also presented to suggest that alterations in PMCA expression in colon cancer cells may provide a growth advantage by promoting proliferation without increasing sensitivity to apoptotic stimuli