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 to Ca ²⁺ -signaling remodeling mediated by Orai1 and Orai3. In ER+ breast cancer cells, Orai3 plays a predominant role in store-operated Ca ²⁺ entry (SOCE), while Orai1 indirectly regulates SOCE and contributes to store-independent Ca ²⁺ 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-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, while Orai1 overexpression attenuated it. Expression of the pore-dead Orai1E106Q mutant in O1KO cells failed to affect Akt phosphorylation, indicating that Ca ²⁺ influx is essential for this modulation. Mechanistically, Orai1 knockout or knockdown attenuated the expression of the adenylyl cyclase 8 (AC8), while Orai1 expression enhanced AC8 levels through a Ca ²⁺ -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.