Ionised calcium is one of the most versatile intracellular second messengers controlling almost all cellular processes including exocytosis, contraction, proliferation, differentiation and apoptosis. The versatility of calcium signalling is mediated through the strict control of intracellular free calcium concentrations. At the resting state cytosolic free calcium ([Ca2+]cyt) is approximately 100 nM while extracellular Ca2+ concentration is 10–20,000 fold higher at around 1-2 mM. Calcium signalling and homeostasis is regulated by the individual and combined effort of a large number of cytosolic and plasma membrane proteins. These proteins belong to different groups such as calcium receptors, calcium binding proteins, calcium channels, calcium pumps, and exchangers. Changes in expression and functionality of one or more of these proteins can cause remodelling of Ca2+ homeostasis and may lead to disease conditions including cancer. In colon cancer, colon epithelial cells are characterized by a lack of differentiation and uncontrolled proliferation. Changes in Ca2+ signalling may have a role in this process as reflected in the altered expression of various calcium channels and pumps in colon cancer including voltage-operated channels, the transient receptor protein (TRP) channel TRPM8 and TRPV6, and sarcoplasmic/endoplasmic reticulum Ca2+-ATPase (SERCA) pumps. In addition, PMCA and SERCA pumps show isoform-specific changes in expression levels during differentiation of colon cancer cell lines. The objective of this thesis was to look into the role of TRP proteins, a major group of calcium channels, in colon cancer differentiation.
Intestinal epithelial cells play a pivotal role in calcium homeostasis by facilitating calcium absorption. TRPV6, a member of the TRP channel family is the major calcium channel responsible for calcium influx into epithelial cells from the intestinal cavity. However, other TRP calcium channels are also expressed in intestinal epithelial cells. In the first part of this thesis, real time RT-PCR was used to assess the mRNA levels of different TRP channels in the HT-29 colon cancer cell line during differentiation. Differentiation was induced using two methods; treatment with the chemical sodium butyrate and culturing post-confluence. Confirmation of differentiation was determined using the mRNA expression of the differentiation marker intestinal alkaline phosphatase (ALPI). A significant increase in TRPV6 mRNA expression was observed with differentiation. The changes in Ca2+ signalling were also assessed using high throughput fluorescence imaging plate reader (FLIPRTETRA) methods. Differentiation was associated with alterations in calcium homeostasis as reflected in differential effects on purinergic and neurotensin-mediated calcium signalling. For example, in undifferentiated HT-29 cells, relative adenosine triphosphate (ATP) and neurotensin (NT) mediated responses on [Ca2+]cyt were similar, whereas induction of differentiation with sodium butyrate (3 mM) was associated with a significant difference between ATP and neurotensin responses. The question arises whether TRPV6 upregulation has a causal role in differentiation or is just a consequence of differentiation. To explore this, TRPV6-overexpressing stable colon cancer cell lines were developed and studied as described in the second part of this thesis.
The functional role of TRPV6 in colon cancer was assessed in TRPV6-overexpressing HT-29 cells for changes in proliferation, differentiation state, and changes in Ca2+ signalling in response to agonist stimulation. Overexpression of TRPV6 did not induce differentiation in HT-29 cells. TRPV6 overexpression also did not exhibit any definitive effect on proliferation rate of HT-29 cells. In the case of ATP and NT-induced changes in relative [Ca2+]cyt, TRPV6-overexpression had no significant effect. However, TRPV6-overexpression did affect store-operated calcium entry (SOCE) and increased basal [Ca2+]cyt influx which may be attributed to the previously reported constitutive activity of TRPV6.
Parallel studies also assessed the consequence of TRPV6 overexpression in the MCF7 breast cancer cell line. Although the TRPV6 channel is involved in calcium transport through mammary epithelial cells and it is overexpressed in a subset of breast cancer cell lines, its role in breast cancer is not fully understood. I assessed TRPV6-overexpressing MCF7 breast cancer cells and did not find any change in cell proliferation compared to controls. There was also no significant change in purinergic receptor-mediated Ca2+ signalling, SOCE and basal [Ca2+]cyt influx. In summary, this thesis describes the assessment of the expression profile of TRP channels during differentiation of HT-29 colon cancer cells. Expression of TRPV6 was increased during differentiation but the overexpression of TRPV6 did not result in significant changes in differentiation and proliferation, but was associated with increases in basal Ca2+ influx. Differentiation of HT-29 cells was also associated with differential changes in purinergic and neurotensin receptor-mediated Ca2+ signalling. The results of this thesis provide further evidence that the differentiation of colon cancer cells is associated with a remodelling of Ca2+ homeostasis.