Isoform-specific modulation of PI3K/Akt1 signaling by Orai channels in estrogen receptor-positive breast cancer

The phosphoinositide 3-kinase (PI3K)/Akt signaling pathway is a fundamental intracellular axis that integrates extracellular stimuli to orchestrate essential cellular processes. The activation of Akt1 represents a key determinant of aggressiveness in estrogen receptor-positive (ER+) breast cancer, a process closely associated with Ca²⁺-signaling remodeling mediated by Orai1 and Orai3. In ER+ breast cancer cells, Orai3 plays a predominant role in store-operated Ca²⁺ entry (SOCE), whereas Orai1 indirectly regulates SOCE and contributes to store-independent Ca²⁺ influx pathways. Here we investigated the regulation of Akt1 activation by Orai3 and Orai1 and characterized the underlying intracellular mechanisms in ER+ breast cancer cells. Our findings reveal distinct, opposing roles for Orai1 and Orai3. Orai3 overexpression in wild-type cells, as well as 2-aminoethoxydiphenyl borate (2-APB)-mediated stimulation of Orai1-knockout (O1KO) MCF-7 cells, enhanced Akt1 phosphorylation, whereas Orai3 knockdown attenuated this effect. This 2-APB-induced activation was impaired by calmodulin-dependent protein kinase kinase (CaMKK) inhibition in a concentration-dependent manner. Conversely, Orai1 deficiency (O1KO) or shRNA-mediated knockdown resulted in enhanced Akt1 phosphorylation, whereas Orai1 overexpression attenuated it. Expression of the pore-dead Orai1E106Q mutant in O1KO cells failed to affect Akt phosphorylation, indicating that Ca²⁺ influx is essential for this modulation. Mechanistically, Orai1 knockout or knockdown attenuated the expression of adenylyl cyclase 8 (AC8), whereas Orai1 expression enhanced AC8 levels through a Ca²⁺-influx-dependent mechanism. Finally, silencing AC8 impaired Orai1-mediated inhibition of Akt phosphorylation. Overall, these findings demonstrate an unprecedented functional antagonism between Orai1 and Orai3 in ER+ breast cancer cells, where distinct signaling pathways mediate their opposing effects on Akt1 phosphorylation.NEW & NOTEWORTHY Orai1 and Orai3 exert opposing effects on the PI3K/Akt1 pathway, revealing a complex regulatory mechanism to coordinate cellular processes, such as survival, in ER+ breast cancer cells. Although Orai3 promotes Akt1 activation via CaMKK to drive tumor progression, Orai1 acts as a modulator on this axis through an AC8/cAMP-dependent mechanism. These findings highlight the functional diversity of Orai proteins and identify them as potential therapeutic targets for modulating Akt1-driven aggressiveness in breast cancer.