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. 2012 Aug 17;287(34):28598-608.
doi: 10.1074/jbc.M112.364737. Epub 2012 Jun 25.

Distinct regulation of cytoplasmic calcium signals and cell death pathways by different plasma membrane calcium ATPase isoforms in MDA-MB-231 breast cancer cells

Merril C Curry  1 , Nicole A Luk, Paraic A Kenny, Sarah J Roberts-Thomson, Gregory R Monteith
Affiliations

Distinct regulation of cytoplasmic calcium signals and cell death pathways by different plasma membrane calcium ATPase isoforms in MDA-MB-231 breast cancer cells

Merril C Curry et al. J Biol Chem. .

Abstract

Plasma membrane calcium ATPases (PMCAs) actively extrude Ca(2+) from the cell and are essential components in maintaining intracellular Ca(2+) homeostasis. There are four PMCA isoforms (PMCA1-4), and alternative splicing of the PMCA genes creates a suite of calcium efflux pumps. The role of these different PMCA isoforms in the control of calcium-regulated cell death pathways and the significance of the expression of multiple isoforms of PMCA in the same cell type are not well understood. In these studies, we assessed the impact of PMCA1 and PMCA4 silencing on cytoplasmic free Ca(2+) signals and cell viability in MDA-MB-231 breast cancer cells. The PMCA1 isoform was the predominant regulator of global Ca(2+) signals in MDA-MB-231 cells. PMCA4 played only a minor role in the regulation of bulk cytosolic Ca(2+), which was more evident at higher Ca(2+) loads. Although PMCA1 or PMCA4 knockdown alone had no effect on MDA-MB-231 cell viability, silencing of these isoforms had distinct consequences on caspase-independent (ionomycin) and -dependent (ABT-263) cell death. PMCA1 knockdown augmented necrosis mediated by the Ca(2+) ionophore ionomycin, whereas apoptosis mediated by the Bcl-2 inhibitor ABT-263 was enhanced by PMCA4 silencing. PMCA4 silencing was also associated with an inhibition of NFκB nuclear translocation, and an NFκB inhibitor phenocopied the effects of PMCA4 silencing in promoting ABT-263-induced cell death. This study demonstrates distinct roles for PMCA1 and PMCA4 in the regulation of calcium signaling and cell death pathways despite the widespread distribution of these two isoforms. The targeting of some PMCA isoforms may enhance the effectiveness of therapies that act through the promotion of cell death pathways in cancer cells.

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Figures

FIGURE 1.
FIGURE 1.
Silencing of PMCA4 and PMCA1 in MDA-MB-231 cells. Quantification of PMCA4 mRNA (A) and PMCA1 mRNA levels (B), 48 h post-siRNA transfection with siPMCA4, siPMCA1, or siNT. Real time RT-PCR data were pooled from nine individual wells, from three independent experiments performed in triplicate. *, p < 0.05, one-tailed Student's t test. C, immunoblots of PMCA4 and total PMCA protein expression, 72 h post-siRNA transfection. PMCA4 (D) and total PMCA (E) protein expression from densitometic analysis normalized to the β-actin loading control, from three independent experiments. *, p < 0.05, one-way ANOVA, Bonferroni post hoc analysis. All data shown are mean ± S.D.
FIGURE 2.
FIGURE 2.
Effect of PMCA1 and PMCA4 silencing on CPA and ATP evoked [Ca2+]CYT increases in MDA-MB-231 breast cancer cells. CPA (10 μm)-mediated (A) and ATP-mediated (100 μm; E) [Ca2+]CYT after transfection with siPMCA1, siPMCA4, or siNT in the presence of extracellular BAPTA (100 μm). Ca2+ traces represent relative mean fluorescence. B and F, time to peak [Ca2+]CYT and rate of [Ca2+]CYT decay after CPA and ATP responses, respectively. C and G, half-peak decay time for CPA and ATP responses, respectively. D and H, area under the curve for CPA and ATP responses, respectively. Bar graphs are mean ± S.D. for Ca2+ transient parameters. All data were pooled from nine individual wells, from three independent experiments performed in triplicate. *, p < 0.05, one-way ANOVA, Bonferroni post hoc analysis. BAPTA, 1,2-bis(o-aminophenoxylethane)-N,N,N′,N′-tetraacetic acid.
FIGURE 3.
FIGURE 3.
Ionomycin-evoked [Ca2+]CYT signals in the presence of PMCA1 and PMCA4 silencing. MDA-MB-231 breast cancer cells were transfected with siPMCA1, siPMCA4, or siNT, and changes in [Ca2+]CYT were assessed after the addition of ionomycin (3 μm (A) or 10 μm (C)). Ca2+ traces represent relative mean fluorescence. B and D, rate of [Ca2+]CYT increase for 3 μm and 10 μm ionomycin Ca2+ responses, respectively. Bar graphs are mean ± S.D. for Ca2+ transient parameters. All data were pooled from nine individual wells, from three independent experiments performed in triplicate. *, p < 0.05, one-way ANOVA, Bonferroni post hoc analysis.
FIGURE 4.
FIGURE 4.
Cell viability in the presence of siRNA-mediated silencing of PMCA1 and PMCA4 gene expression. MDA-MB-231 breast cancer cells were transfected with siPMCA4, siPMCA1, or siNT, incubated for a further 48 h and then assessed for cell viability. Cell viability for siNT (A), siPMCA4 (B), and siPMCA1 (C) presented as dot plots of Hoechst 33342 and propidium iodide fluorescence. Each dot plot shows an equal cell number (10,000 cells) randomly selected from three independent experiments performed in triplicate wells. D, bar graph showing the mean ± S.D. of the proportion of viable cells 120 h post-siRNA transfection from three independent experiments. *, p > 0.05, one-way ANOVA, Bonferroni post hoc analysis. PMCA4 mRNA (E) and PMCA1 mRNA (F) levels, 120 h post-siRNA transfection with siPMCA4, siPMCA1, or siNT. Real time RT-PCR data were pooled from six individual wells, from three independent experiments performed in duplicate. *, p < 0.05, one-tailed Student's t test.
FIGURE 5.
FIGURE 5.
Effects of the caspase inhibitor Z-VAD-FMK on ionomycin and ABT-263-mediated cell death in MDA-MB-231 breast cancer cells. A–F, dot plots for control, ionomycin, or ABT-263 either in the absence or presence of Z-VAD-FMK. Each dot plot represents an equal number of total cells (10,000 cells) selected at random from three independent experiments. G and H, the pooled data for the effect of Z-VAD-FMK on the proportion of dead cells induced by ionomycin or ABT-263 (mean ± S.D.) from three independent experiments. *, p < 0.05, repeated measures two-way ANOVA, Bonferroni post hoc analysis.
FIGURE 6.
FIGURE 6.
PMCA1 and PMCA4 silencing effects in promoting ionomycin-mediated cell death in MDA-MB-231 breast cancer cells. Dot plots of Hoechst 33342 and propidium iodide fluorescence in cells transfected with siNT (A and D), siPMCA4 (B and E), or siPMCA1 (C and F) following treatment with 3 μm or 10 μm ionomycin. Each dot plot represents an equal number of total cells (10,000 cells) selected at random from three independent experiments. G, the proportion of necrotic cells pooled from three independent experiments (mean ± S.D.). *, p < 0.05, repeated measures two-way ANOVA, Bonferroni post hoc analysis.
FIGURE 7.
FIGURE 7.
PMCA1 and PMCA4 silencing effects in promoting ABT-263-mediated cell death in MDA-MB-231 breast cancer cells. Dot plots of Hoechst 33342 and propidium iodide florescence in cells transfected with siNT (A and D), siPMCA4 (B and E), or siPMCA1 (C and F) following treatment with ABT-263 (ABT). Each dot plot represents an equal number of total cells (10,000 cells) selected at random from three independent experiments. G, the proportion of apoptotic cells pooled from three independent experiments (mean ± S.D.). *, p < 0.05, repeated measures two-way ANOVA, Bonferroni post hoc analysis.
FIGURE 8.
FIGURE 8.
NFκB activity in the presence of PMCA1 and PMCA4 siRNA and the effect of pharmacological inhibition of NFκB on ABT-263-mediated cell death. A, MDA-MB-231 breast cancer cells stained for NFκB (orange) with DAPI (nuclei, blue) and with white arrows depicting translocation of NFκB to the nucleus in the (i) absence of PMA (50 nm), (ii) presence of PMA (50 nm), (iii) presence of siPMCA1 and PMA (50 nm), and (iv) presence of siPMCA4 and PMA (50 nm). B, percentage of cells with NFκB nuclear translocation in siRNA transfected cells following the addition of PMA (50 nm) normalized to the dimethyl sulfoxide control. C, the proportion of viable cells following IMD-0354 and/or ABT-263 (ABT) treatment. D, the proportion of apoptotic cells following IMD-0354 and/or ABT-263 treatment. E–I, dot plots of Hoechst 33342 and propidium iodide fluorescence in cells transfected with siNT or with siPMCA4 in the presence of IMD-0354 and/or ABT-263. Each dot plot represents an equal number of total cells (10,000 cells) selected at random from three independent experiments. All data were from three independent experiments and are presented as mean ± S.D. where relevant. *, p < 0.05, repeated measures two-way ANOVA, Bonferroni post hoc analysis. Scale bar, 25 μm.
FIGURE 9.
FIGURE 9.
The expression of PMCA1 and PMCA4 in breast cancer cell lines and clinical samples. A, the relative expression level (log2) of PMCA1 was assessed in a microarray data set consisting of luminal (n = 13) and basal (n = 10) breast cancer cell lines grown in three-dimensional culture. B, relative PMCA4 expression levels in these breast cancer cell lines. C, the relative expression level of PMCA1 was assessed in a microarray data set consisting of estrogen receptor-negative (n = 77) and -positive (n = 209) breast tumors. D, the relative expression level of PMCA4 was assessed in the same breast tumor data set. A bar indicates the median, and significance was evaluated using the Mann-Whitney test. *, p < 0.05. E, Kaplan-Meier analysis of relapse-free survival of the breast cancer patients stratified by their tumor levels of PMCA1. F, relapse-free survival of breast cancer patients stratified by their tumor levels of PMCA4. 3D, three-dimensional.
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