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      Non-Stimulated, Agonist-Stimulated and Store-Operated Ca 2+ Influx in MDA-MB-468 Breast Cancer Cells and the Effect of EGF-Induced EMT on Calcium Entry

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          Abstract

          In addition to their well-defined roles in replenishing depleted endoplasmic reticulum (ER) Ca 2+ reserves, molecular components of the store-operated Ca 2+ 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 Ca 2+ influx, and that MDA-MB-468 cells prior to EMT induction have a high level of non-stimulated Ca 2+ influx. The potential roles for specific Ca 2+ 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 Ca 2+ influx were significantly inhibited with ORAI1 silencing. TRPC1 knockdown attenuated non-stimulated Ca 2+ influx in a manner dependent on Ca 2+ influx via ORAI1. TRPC1 silencing was also associated with reduced ERK1/2 phosphorylation and changes in the rate of Ca 2+ release from the ER associated with the inhibition of the sarco/endoplasmic reticulum Ca 2+-ATPase (time to peak [Ca 2+] CYT = 188.7±34.6 s (TRPC1 siRNA) versus 124.0±9.5 s (non-targeting siRNA); P<0.05). These studies indicate that EMT in MDA-MB-468 breast cancer cells is associated with a pronounced remodeling of Ca 2+ 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 Ca 2+ influx in non-stimulated cells.

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          Most cited references 48

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          STIM is a Ca2+ sensor essential for Ca2+-store-depletion-triggered Ca2+ influx.

          Ca(2+) signaling in nonexcitable cells is typically initiated by receptor-triggered production of inositol-1,4,5-trisphosphate and the release of Ca(2+) from intracellular stores. An elusive signaling process senses the Ca(2+) store depletion and triggers the opening of plasma membrane Ca(2+) channels. The resulting sustained Ca(2+) signals are required for many physiological responses, such as T cell activation and differentiation. Here, we monitored receptor-triggered Ca(2+) signals in cells transfected with siRNAs against 2,304 human signaling proteins, and we identified two proteins required for Ca(2+)-store-depletion-mediated Ca(2+) influx, STIM1 and STIM2. These proteins have a single transmembrane region with a putative Ca(2+) binding domain in the lumen of the endoplasmic reticulum. Ca(2+) store depletion led to a rapid translocation of STIM1 into puncta that accumulated near the plasma membrane. Introducing a point mutation in the STIM1 Ca(2+) binding domain resulted in prelocalization of the protein in puncta, and this mutant failed to respond to store depletion. Our study suggests that STIM proteins function as Ca(2+) store sensors in the signaling pathway connecting Ca(2+) store depletion to Ca(2+) influx.
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            A mutation in Orai1 causes immune deficiency by abrogating CRAC channel function.

            Antigen stimulation of immune cells triggers Ca2+ entry through Ca2+ release-activated Ca2+ (CRAC) channels, promoting the immune response to pathogens by activating the transcription factor NFAT. We have previously shown that cells from patients with one form of hereditary severe combined immune deficiency (SCID) syndrome are defective in store-operated Ca2+ entry and CRAC channel function. Here we identify the genetic defect in these patients, using a combination of two unbiased genome-wide approaches: a modified linkage analysis with single-nucleotide polymorphism arrays, and a Drosophila RNA interference screen designed to identify regulators of store-operated Ca2+ entry and NFAT nuclear import. Both approaches converged on a novel protein that we call Orai1, which contains four putative transmembrane segments. The SCID patients are homozygous for a single missense mutation in ORAI1, and expression of wild-type Orai1 in SCID T cells restores store-operated Ca2+ influx and the CRAC current (I(CRAC)). We propose that Orai1 is an essential component or regulator of the CRAC channel complex.
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              CRACM1 is a plasma membrane protein essential for store-operated Ca2+ entry.

              Store-operated Ca2+ entry is mediated by Ca2+ release-activated Ca2+ (CRAC) channels following Ca2+ release from intracellular stores. We performed a genome-wide RNA interference (RNAi) screen in Drosophila cells to identify proteins that inhibit store-operated Ca2+ influx. A secondary patch-clamp screen identified CRACM1 and CRACM2 (CRAC modulators 1 and 2) as modulators of Drosophila CRAC currents. We characterized the human ortholog of CRACM1, a plasma membrane-resident protein encoded by gene FLJ14466. Although overexpression of CRACM1 did not affect CRAC currents, RNAi-mediated knockdown disrupted its activation. CRACM1 could be the CRAC channel itself, a subunit of it, or a component of the CRAC signaling machinery.
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                Author and article information

                Contributors
                Role: Editor
                Journal
                PLoS One
                PLoS ONE
                plos
                plosone
                PLoS ONE
                Public Library of Science (San Francisco, USA )
                1932-6203
                2012
                30 May 2012
                : 7
                : 5
                Affiliations
                School of Pharmacy, The University of Queensland, Brisbane, Queensland, Australia
                University of Queensland, Australia
                Author notes

                Conceived and designed the experiments: FD GM SRT DG AP MOP. Performed the experiments: FD AP DG. Analyzed the data: FD AP PC GM. Contributed reagents/materials/analysis tools: GM SRT MOP. Wrote the paper: FD GM. Edited the manuscript: SRT DG AP MOP PC.

                Article
                PONE-D-12-07723
                10.1371/journal.pone.0036923
                3364242
                22666335
                Davis et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
                Page count
                Pages: 11
                Categories
                Research Article
                Biology
                Genetics
                Cancer Genetics
                Gene Function
                Molecular Cell Biology
                Signal Transduction
                Signaling Cascades
                Calcium Signaling Cascade
                Signaling in Cellular Processes
                Calcium Signaling
                Signaling Pathways
                Calcium-Mediated Signal Transduction
                Medicine
                Obstetrics and Gynecology
                Breast Cancer
                Oncology
                Basic Cancer Research
                Metastasis

                Uncategorized

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