Calcium signalling modifying proteins as targets for breast cancer therapy

Abstract

The calcium ion (Ca2+) is a ubiquitous second messenger that controls intracellular signals that trigger various cellular responses including cell proliferation, gene transcription and cell death. Intracellular Ca2+ signalling is tightly regulated by the actions of Ca2+ -transporting proteins including Ca2+ channels, Ca2+ exchangers, Ca2+ -ATPases and their regulators. Evidence suggests that deregulation of Ca2+ signalling is associated with several pathophysiological conditions including cancer. Altered expression and/or activity of some Ca2+ transporters have been demonstrated to occur during cancer development and progression. Thus, Ca2+ transporters represent attractive drug targets for therapeutic intervention. The work in this thesis is centred on identifying proteins implicated in Ca2+ signalling that represent drug targets for breast cancer treatment. This thesis also performed functional assessment of such targets in the regulation of Ca2+ -dependent processes including cell proliferation and cell death in breast cancer cells. The first part of this thesis addressed the utility of high throughput siRNA screening in combination with high content imaging in a human basal-like breast cancer cell line for the identification of potential therapeutic targets which are involved in Ca2+ signalling. Screening of an siRNA-based library against Ca2+ signalling modifying proteins revealed 39 possible hits based on the pronounced effects on human MDA-MB-231 basal-like breast cancer cells compared to nontumorigenic MCF-10A cells. From these hits, eight candidate genes were advanced to target validation based on target druggability, plasma membrane localisation and a lack of previous assessment in breast cancer cells. These specific selected targets were then assessed in basal breast cancer cell lines through the assessment of their mRNA levels and the consequences of their silencing and/or pharmacological modulation on Ca2+ signalling, cell viability and cell death.Of the eight candidate genes, three hits were undetectable by real-time RT-PCR in MDA-MB-231 cells. CD24 was the first selected candidate gene and was characterised in the human basal-like breast cancer cell lines MDA-MB-231 and MDA-MB-468. CD24 mRNA levels were higher (more than 800-fold) in MDA-MB-468 compared to MDA-MB-231 cells. Since CD24 has also been linked to the chemokine receptor CXCR4 in breast cancer, the functional role of CD24 was characterised by examining its effect on CXCL12/CXCR4-mediated Ca2+ signalling. Highly metastatic MDA-MB-231 cells displayed more pronounced Ca2+ responses when stimulated with CXCL12 (CXCR4 receptor ligand) than poorly metastatic MDA-MB-468 cells. CD24 silencing in MDA-MB-468 cells did not alter the nature of ATP-induced Ca2+ transients and did not promote an increase in cytosolic free Ca2+ ([Ca2+]CYT). Real-time RT-PCR showed that CXCR4 expression was higher in MDA-MB-231 than MDA-MB-468 cells. Hence, CXCR4 but not CD24 levels are likely responsible for the differences in CXCL12 responsiveness between these two cell lines. CXCR4 expression was significantly reduced during epidermal growth factor (EGF)-induced epithelial to mesenchymal transition (EMT) in MDA-MB-468 cells, indicating that increased CXCR4 is not associated with the acquisition of a more mesenchymal phenotype.This thesis then explored the functional role of another selected target, the Ca2+ permeable ion channel TRPV4 in human basal-like breast cancer cells. Real-time RT-PCR analysis revealed that TRPV4 was present in both MDA-MB-231 and MDA-MB-468 cells, with higher levels (approximately 30-fold) in MDA-MB-468 cells. The functional activity of TRPV4 channels was then investigated using a known TRPV4 agonist - GSK1016790A. Consistent with the levels of TRPV4 mRNA, TRPV4 activation by GSK1016790A elicited more pronounced increases in [Ca2+]CYT in MDA-MB-468 compared to MDA-MB-231 cells. GSK1016790A concentrationresponse curves revealed a lower EC50 value in MDA-MB-468 cells than MDA-MB-231 cells (3.9 nM and 76 nM, respectively). Silencing of TRPV4 using siRNA significantly reduced GSK1016790A-induced Ca2+ influx in both cell lines, indicating that TRPV4 is a functional Ca2+ entry channel in both MDA-MB-231 and MDA-MB-468 cells.Pharmacological inhibition of TRPV4 channels using the TRPV4 antagonist, RN 1734 had no significant effect on the viable cell number in either MDA-MB-231 or MDA-MB-468 breast cancer cells. However, pharmacological activation of TRPV4 channels using GSK1016790A in MDAMB-468 cells but not in MDA-MB-231 cells led to a concentration-dependent induction of cell death. GSK1016790A-mediated cell death was significantly attenuated in the presence of TRPV4 siRNA. The mechanism of GSK1016790A-induced cell death in MDA-MB-468 cells was independent of caspase, as indicated by the insensitivity to cell death by the caspase inhibitor ZVAD-FMK. The work outlined in this thesis demonstrates the importance of confirmation experiments for targets identified by high throughput siRNA screening. These studies have also identified TRPV4 pharmacological activation as a potential novel mechanism to induce the death of breast cancer cells that overexpress this Ca2+ permeable ion channel.