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Adenylyl Cyclase Type 8 Overexpression Impairs Phosphorylation-Dependent Orai1 Inactivation and Promotes Migration in MDA-MB-231 Breast Cancer Cells

1
Department of Physiology, (Cellular Physiology Research Group), Institute of Molecular Pathology Biomarkers, University of Extremadura, 10003 Caceres, Spain
2
Department of Physiology, (Smooth Muscle Physiology Research Group), Institute of Molecular Pathology Biomarkers, University of Extremadura, 10003 Caceres, Spain
3
Department of Medical Physiology and Biophysics, Institute of Biomedicine of Sevilla, 41013 Sevilla, Spain
*
Authors to whom correspondence should be addressed.
These authors contributed equally to this work.
Cancers 2019, 11(11), 1624; https://doi.org/10.3390/cancers11111624
Received: 10 October 2019 / Accepted: 21 October 2019 / Published: 23 October 2019
(This article belongs to the Special Issue Targeting Calcium Signaling in Cancer Cells)
Orai1 plays a major role in store-operated Ca2+ entry (SOCE) in triple-negative breast cancer (TNBC) cells. This channel is inactivated via different mechanisms, including protein kinase C (PKC) and protein kinase A (PKA)-dependent phosphorylation at Ser-27 and Ser-30 or Ser-34, respectively, which shapes the Ca2+ responses to agonists. The Ca2+ calmodulin-activated adenylyl cyclase type 8 (AC8) was reported to interact directly with Orai1, thus mediating a dynamic interplay between the Ca2+- and cyclic adenosine monophosphate (cAMP)-dependent signaling pathways. Here, we show that the breast cancer cell lines MCF7 and MDA-MB-231 exhibit enhanced expression of Orai1 and AC8 as compared to the non-tumoral breast epithelial MCF10A cell line. In these cells, AC8 interacts with the Orai1α variant in a manner that is not regulated by Orai1 phosphorylation. AC8 knockdown in MDA-MB-231 cells, using two different small interfering RNAs (siRNAs), attenuates thapsigargin (TG)-induced Ca2+ entry and also Ca2+ influx mediated by co-expression of Orai1 and the Orai1-activating small fragment (OASF) of STIM1 (stromal interaction molecule-1). Conversely, AC8 overexpression enhances SOCE, as well as Ca2+ entry, in cells co-expressing Orai1 and OASF. In MDA-MB-231 cells, we found that AC8 overexpression reduces the Orai1 phosphoserine content, thus suggesting that AC8 interferes with Orai1 serine phosphorylation, which takes place at residues located in the AC8-binding site. Consistent with this, the subset of Orai1 associated with AC8 in naïve MDA-MB-231 cells is not phosphorylated in serine residues in contrast to the AC8-independent Orai1 subset. AC8 expression knockdown attenuates migration of MCF7 and MDA-MB-231 cells, while this maneuver has no effect in the MCF10A cell line, which is likely attributed to the low expression of AC8 in these cells. We found that AC8 is required for FAK (focal adhesion kinase) phosphorylation in MDA-MB-231 cells, which might explain its role in cell migration. Finally, we found that AC8 is required for TNBC cell proliferation. These findings indicate that overexpression of AC8 in breast cancer MDA-MB-231 cells impairs the phosphorylation-dependent Orai1 inactivation, a mechanism that might support the enhanced ability of these cells to migrate. View Full-Text
Keywords: orai1α; adenylyl cyclase 8; store-operated calcium entry; breast cancer cells; migration orai1α; adenylyl cyclase 8; store-operated calcium entry; breast cancer cells; migration
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MDPI and ACS Style

Sanchez-Collado, J.; Lopez, J.J.; Jardin, I.; Camello, P.J.; Falcon, D.; Regodon, S.; Salido, G.M.; Smani, T.; Rosado, J.A. Adenylyl Cyclase Type 8 Overexpression Impairs Phosphorylation-Dependent Orai1 Inactivation and Promotes Migration in MDA-MB-231 Breast Cancer Cells. Cancers 2019, 11, 1624. https://doi.org/10.3390/cancers11111624

AMA Style

Sanchez-Collado J, Lopez JJ, Jardin I, Camello PJ, Falcon D, Regodon S, Salido GM, Smani T, Rosado JA. Adenylyl Cyclase Type 8 Overexpression Impairs Phosphorylation-Dependent Orai1 Inactivation and Promotes Migration in MDA-MB-231 Breast Cancer Cells. Cancers. 2019; 11(11):1624. https://doi.org/10.3390/cancers11111624

Chicago/Turabian Style

Sanchez-Collado, Jose; Lopez, Jose J.; Jardin, Isaac; Camello, Pedro J.; Falcon, Debora; Regodon, Sergio; Salido, Gines M.; Smani, Tarik; Rosado, Juan A. 2019. "Adenylyl Cyclase Type 8 Overexpression Impairs Phosphorylation-Dependent Orai1 Inactivation and Promotes Migration in MDA-MB-231 Breast Cancer Cells" Cancers 11, no. 11: 1624. https://doi.org/10.3390/cancers11111624

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