Adenylyl Cyclase Type 8 Overexpression Impairs Phosphorylation-Dependent Orai1 Inactivation and Promotes Migration in MDA-MB-231 Breast Cancer Cells
Adenylyl Cyclase Type 8 Overexpression Impairs Phosphorylation-Dependent Orai1 Inactivation and Promotes Migration in MDA-MB-231 Breast Cancer Cells
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DOI：
10.3390/cancers11111624
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发表时间：
2019-11-01
期刊：
影响因子：
5.2
通讯作者：
Rosado, Juan A.
中科院分区：
文献类型：
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作者：
Sanchez-Collado, Jose;Lopez, Jose J.;Rosado, Juan A.
Orai1 在三阴性乳腺癌 (TNBC) 细胞中钙池操纵的 Ca2+ 内流 (SOCE) 中发挥重要作用。该通道通过不同的机制失活，包括分别在 Ser-27 和 Ser-30 或 Ser-34 处依赖蛋白激酶 C (PKC) 和蛋白激酶 A (PKA) 的磷酸化，从而形成 Ca2+ 对激动剂的反应。据报道，Ca2+钙调蛋白激活的腺苷酸环化酶8型（AC8）直接与Orai1相互作用，从而介导Ca2+-和环磷酸腺苷（cAMP）依赖性信号通路之间的动态相互作用。在这里，我们发现与非肿瘤乳腺上皮细胞系 MCF10A 相比，乳腺癌细胞系 MCF7 和 MDA-MB-231 表现出 Orai1 和 AC8 的表达增强。在这些细胞中，AC8 与 Orai1 α 变体以不受 Orai1 磷酸化调节的方式相互作用。使用两种不同的小干扰 RNA (siRNA) 敲低 MDA-MB-231 细胞中的 AC8，可减弱毒胡萝卜素 (TG) 诱导的 Ca2+ 进入以及由 Orai1 和 STIM1（基质相互作用分子-1）的 Orai1 激活小片段 (OASF) 共表达介导的 Ca2+ 流入。相反，在共表达 Orai1 和 OASF 的细胞中，AC8 过表达可增强 SOCE 以及 Ca2+ 进入。在MDA-MB-231细胞中，我们发现AC8过表达降低了Orai1磷酸丝氨酸含量，因此表明AC8干扰Orai1丝氨酸磷酸化，该磷酸化发生在位于AC8结合位点的残基处。与此一致的是，与 AC8 独立的 Orai1 子集相比，在初始 MDA-MB-231 细胞中与 AC8 相关的 Orai1 子集的丝氨酸残基并未磷酸化。 AC8 表达敲低会减弱 MCF7 和 MDA-MB-231 细胞的迁移，而这种操作对 MCF10A 细胞系没有影响，这可能归因于这些细胞中 AC8 的低表达。我们发现AC8是MDA-MB-231细胞中FAK（粘着斑激酶）磷酸化所必需的，这可能解释了它在细胞迁移中的作用。最后，我们发现AC8是TNBC细胞增殖所必需的。这些发现表明，乳腺癌 MDA-MB-231 细胞中 AC8 的过度表达会损害磷酸化依赖性的 Orai1 失活，这种机制可能支持这些细胞迁移能力的增强。
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 Orai1 alpha 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 naive 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.