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

Jose Sanchez-Collado; Jose J Lopez; Isaac Jardin; Pedro J Camello; Debora Falcon; Sergio Regodon; Gines M Salido; Tarik Smani; Juan A Rosado

doi:10.3390/cancers11111624

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

Cancers (Basel). 2019 Oct 23;11(11):1624. doi: 10.3390/cancers11111624.

Authors

Jose Sanchez-Collado¹, Jose J Lopez², Isaac Jardin³, Pedro J Camello⁴, Debora Falcon⁵, Sergio Regodon⁶, Gines M Salido⁷, Tarik Smani⁸, Juan A Rosado⁹

Affiliations

¹ Department of Physiology, (Cellular Physiology Research Group), Institute of Molecular Pathology Biomarkers, University of Extremadura, 10003 Caceres, Spain. josesc@unex.es.
² Department of Physiology, (Cellular Physiology Research Group), Institute of Molecular Pathology Biomarkers, University of Extremadura, 10003 Caceres, Spain. jjlopez@unex.es.
³ Department of Physiology, (Cellular Physiology Research Group), Institute of Molecular Pathology Biomarkers, University of Extremadura, 10003 Caceres, Spain. ijp@unex.es.
⁴ Department of Physiology, (Smooth Muscle Physiology Research Group), Institute of Molecular Pathology Biomarkers, University of Extremadura, 10003 Caceres, Spain. pcamello@unex.es.
⁵ Department of Medical Physiology and Biophysics, Institute of Biomedicine of Sevilla, 41013 Sevilla, Spain. dfalboy@gmail.com.
⁶ Department of Physiology, (Cellular Physiology Research Group), Institute of Molecular Pathology Biomarkers, University of Extremadura, 10003 Caceres, Spain. sergio@unex.es.
⁷ Department of Physiology, (Cellular Physiology Research Group), Institute of Molecular Pathology Biomarkers, University of Extremadura, 10003 Caceres, Spain. gsalido@unex.es.
⁸ Department of Medical Physiology and Biophysics, Institute of Biomedicine of Sevilla, 41013 Sevilla, Spain. tasmani@us.es.
⁹ Department of Physiology, (Cellular Physiology Research Group), Institute of Molecular Pathology Biomarkers, University of Extremadura, 10003 Caceres, Spain. jarosado@unex.es.

Abstract

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.

Keywords: adenylyl cyclase 8; breast cancer cells; migration; orai1α; store-operated calcium entry.