Abstract A30: Inhibition of PMCA4 as a strategy to increase the effectiveness of Bcl-2 inhibitor-mediated cell death in breast cancer cells.

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Abstract Intracellular calcium is a vital cell signaling molecule that can influence a myriad of important cellular functions, including those deregulated in the cancer cell. Consequently Ca2+ levels within the cell must be precisely controlled. Amongst the suite of proteins controlling calcium homeostasis are the plasma membrane calcium ATPases (PMCAs), which belong to a family of druggable proteins. PMCAs are pumps that reside on the plasma membrane and are responsible for the efflux of intracellular Ca2+ and thus are important in maintaining basal cellular calcium levels. Sustained increases in intracellular Ca2+ can be a trigger for cell death and agents that induce alterations in calcium signaling could be a strategy to restore the sensitivity of a cancer cell to death stimuli. The aim of this study was to explore the effectiveness of inhibiting two PMCA isoforms, PMCA1 and PMCA4, on the augmentation of cell death initiated by a Ca2+ionophore (ionomycin) and a Bcl-2 inhibitor (ABT-263) in MDA-MB-231 breast cancer cells. Ionomycin represents a caspase-independent mechanism of cell death and ABT-263 enhances cell death via a caspase-dependent mechanism. PMCA1 and PMCA4 were inhibited using siRNA and the effect on cell death was measured by staining with Hoechst 33342 and propidium iodide using high content imaging (ImageXpress). Intracellular Ca2+ was determined using a FLIPRTETRA with cells loaded with the calcium sensor Fluo4-AM. Inhibition of PMCA1 was associated with significant changes in global cytosolic free Ca2+ induced by purinergic receptor activation, whereas the same effect was not seen with PMCA4 inhibition. Consistent with effects on global calcium levels, ionomycin (3 μM)-induced cell death was significantly augmented by PMCA1 but not PMCA4 siRNA. However, ABT-263 (3 μM)-induced cell death was augmented only by inhibition of PMCA4, suggesting that PMCA4 siRNA-mediated inhibition of ABT-263 apoptosis is mediated through effects on local and/or specific calcium signaling processes. PMCA4 can alter the activity of calcium-dependent transcription factors in cardiac myocytes independent of global cytosolic calcium. In MDA-MB-231 cells, the effects of PMCA4 inhibition appear to be associated with alterations in the activity of NFkappaB. These results highlight the specificity of different isoforms of PMCA in the modulation of cell death pathways in MDA-MB-231 breast cancer cells. Inhibition of PMCA4 may be a viable adjuvant therapy when combined with Bcl-2 family inhibitors for the treatment of breast cancer. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2011 Nov 12-16; San Francisco, CA. Philadelphia (PA): AACR; Mol Cancer Ther 2011;10(11 Suppl):Abstract nr A30.