Non-Stimulated, Agonist-Stimulated and Store-Operated Ca²⁺ Influx in MDA-MB-468 Breast Cancer Cells and the Effect of EGF-Induced EMT on Calcium Entry

<div><p>In addition to their well-defined roles in replenishing depleted endoplasmic reticulum (ER) Ca²⁺ reserves, molecular components of the store-operated Ca²⁺ entry pathway regulate breast cancer metastasis. A process implicated in cancer metastasis that describes the conversion to a more invasive phenotype is epithelial-mesenchymal transition (EMT). In this study we show that EGF-induced EMT in MDA-MB-468 breast cancer cells is associated with a reduction in agonist-stimulated and store-operated Ca²⁺ influx, and that MDA-MB-468 cells prior to EMT induction have a high level of non-stimulated Ca²⁺ influx. The potential roles for specific Ca²⁺ channels in these pathways were assessed by siRNA-mediated silencing of ORAI1 and transient receptor potential canonical type 1 (TRPC1) channels in MDA-MB-468 breast cancer cells. Non-stimulated, agonist-stimulated and store-operated Ca²⁺ influx were significantly inhibited with ORAI1 silencing. TRPC1 knockdown attenuated non-stimulated Ca²⁺ influx in a manner dependent on Ca²⁺ influx via ORAI1. TRPC1 silencing was also associated with reduced ERK1/2 phosphorylation and changes in the rate of Ca²⁺ release from the ER associated with the inhibition of the sarco/endoplasmic reticulum Ca²⁺-ATPase (time to peak [Ca²⁺]_CYT = 188.7±34.6 s (TRPC1 siRNA) versus 124.0±9.5 s (non-targeting siRNA); <em>P</em><0.05). These studies indicate that EMT in MDA-MB-468 breast cancer cells is associated with a pronounced remodeling of Ca²⁺ influx, which may be due to altered ORAI1 and/or TRPC1 channel function. Our findings also suggest that TRPC1 channels in MDA-MB-468 cells contribute to ORAI1-mediated Ca²⁺ influx in non-stimulated cells.</p> </div

mRNA levels of TRPC1 and ORAI1 with EGF-induced EMT.

<p>TRPC1 (<b>A</b>) and ORAI1 (<b>B</b>) mRNA levels in MDA-MB-468 breast cancer cells stimulated with EGF to induce EMT. Values show mean ± S.D. for nine wells from three independent experiments; * <i>P</i><0.05 (unpaired t-test).</p

siRNA-mediated silencing of ORAI1 and TRPC1 in MDA-MB-468 breast cancer cells.

<p><b>A</b>) Quantitation of TRPC1 mRNA expression 48 h after treatment with TRPC1 siRNA (siTRPC1) relative to the non-targeting siRNA control (siNT). <b>B</b>) Assessment of ORAI1 expression 48 h post transfection with ORAI1 siRNA (siORAI1). Values show mean ± S.D. for six wells from three independent experiments; * <i>P</i><0.05 (unpaired t-test).</p

Effect of TRPC1 and ORAI1 silencing on constitutive ERK1/2 activity and cell proliferation.

<p><b>A</b>) Representative immunoblot showing constitutive phosphorylation and total expression of ERK1/2 in MDA-MB-468 cells with ORAI1 or TRPC1 silencing. <b>B</b>) Densitometric data was obtained from the pooled data (three independent immunoblots) and shows ERK1/2 phosphorylation relative to total ERK1/2 expression; * <i>P</i><0.05 (one-way ANOVA with Bonferroni post-tests). <b>C</b>) DAPI (cell number) and EdU staining (showing cells in S-phase of the cell cycle). <b>D</b>) Quantitation of the average cell count and (<b>E</b>) EdU positivity for nine wells from three independent experiments.* <i>P</i><0.05 (unpaired t-test). All graphs show mean ± S.D. Scale bar represents 100 µm.</p

Assessment of cytosolic Ca²⁺ signaling in MDA-MB-468 and MDA-MB-231 breast cancer cell lines.

<p>Average ATP-stimulated Ca²⁺ entry response (100 µM ATP), store-operated Ca²⁺ entry response (10 µM CPA) and non-stimulated Ca²⁺ influx (DMSO control) were assessed in <b>A</b>) MDA-MB-468 and <b>B</b>) MDA-MB-231 cells. Quantitation of peak relative Ca²⁺ influx (peak 2) in <b>C</b>) MDA-MB-468 and <b>D</b>) MDA-MD-231 cells; shown as average ± S.D. (n = 9 for MDA-MB-468 & n = 4 for MDA-MB-231 cells). <b>E</b>) MDA-MB-468 cells exhibit spontaneous and asynchronous Ca²⁺ oscillations that are attenuated by extracellular Ca²⁺ chelation (BAPTA). <b>F</b>) Still image showing the Y-plane section and <b>G</b>) the Y-plane projection through time. See also <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0036923#pone.0036923.s001" target="_blank">Movie S1</a>. Representative of five movies from three independent experiments.</p

Non-stimulated Ca²⁺ influx in MDA-MB-468 cells induced to undergo EMT with EGF.

<p>MDA-MB-468 breast cancer cells stimulated with EGF to induce EMT (MDA-MB-468-EMT) have elevated transcription of the mesenchymal markers <b>A</b>) Twist, <b>B</b>) Snail, <b>C</b>) fibronectin and <b>D</b>) vimentin. MDA-MB-468-EMT cells show reduced non-stimulated Ca²⁺ influx, shown as <b>E</b>) average cytosolic Ca²⁺ response, <b>F</b>) peak relative Ca²⁺ influx (peak 2) and <b>G</b>) ratio of Ca²⁺ influx amplitude (peak 2) divided by AUC (t = 0–705 s). Values show mean ± S.D. for nine wells from three independent experiments. * <i>P</i><0.05 (unpaired t-test).</p

Summary of changes in Ca²⁺ homeostasis with EGF-induced EMT and silencing of ORAI1 (siORAI1) or TRPC1 (siTRPC1).

<p>Arrows signify whether the epithelial, mesenchymal or knockdown phenotypes are associated with a significant increase (↑) or decrease (↓) or no change (↔) in Ca²⁺ influx via each pathway. Changes in the release of ER Ca²⁺ upon SERCA inhibition are described as fast, slow or unchanged (↔) for each condition.</p

Agonist-stimulated and store-operated Ca²⁺ entry in MDA-MB-468 cells with EGF-induced EMT.

<p>Average cytosolic Ca²⁺ response, peak relative Ca²⁺ influx (peak 2) and ratio of Ca²⁺ influx amplitude (peak 2) divided by AUC (t = 0–705 s) mediated by 100 µM ATP (<b>A–C</b>), 30 nM trypsin (<b>D–F</b>) and 10 µM CPA (<b>G–I</b>). Average time to peak (t = 0–705 s) for <b>J</b>) ATP, <b>K</b>) trypsin and <b>L</b>) CPA. Values show mean ± S.D. for nine wells from three independent experiments; * <i>P</i><0.05 (unpaired t-test).</p

Altered ER Ca²⁺ release kinetics with TRPC1 silencing.

<p>Quantitation of the time to reach peak cytosolic Ca²⁺ (t = 0–705 s) with CPA in MDA-MB-468 cells with ORAI1 or TRPC1 silencing. Values show mean ± S.D. for nine wells from three independent experiments; * <i>P</i><0.05 (one-way ANOVA with Bonferroni post-tests).</p

Effect of TRPC1 and ORAI1 silencing on Ca²⁺ influx pathways in MDA-MB-468 breast cancer cells.

<p>Non-stimulated Ca²⁺ influx was assessed in MDA-MB-468 breast cancer cells with ORAI1 silencing (siORAI1) or TRPC1 silencing (siTRPC1); <b>A</b>) average cytosolic Ca²⁺ response, <b>B</b>) peak relative Ca²⁺ influx (peak 2) and <b>C</b>) ratio of Ca²⁺ influx amplitude (peak 2) divided by AUC (t = 0-705 s). The role of ORAI1 and TRPC1 in agonist-stimulated Ca²⁺ entry, mediated by 100 µM ATP (<b>D–F</b>) and 30 nM trypsin (<b>G–I</b>), and store-operated Ca²⁺ entry with 10 µM CPA (<b>J–L</b>) was assessed. Data show mean ± S.D. for nine wells from three independent experiments; * <i>P</i><0.05 (one-way ANOVA with Bonferroni post-tests).</p