Orai3 is an estrogen receptor α‐regulated Ca ²⁺ channel that promotes tumorigenesis

Abstract

Store-operated Ca(2+) entry (SOCE) encoded by Orai1 proteins is a ubiquitous Ca(2+)-selective conductance involved in cellular proliferation and migration. We recently described up-regulation of Orai3 channels that selectively mediate SOCE in estrogen receptor α-expressing (ERα(+)) breast cancer cells. However, the connection between ERα and Orai3 and the role of Orai3 in tumorigenesis remain unknown. Here, we show that ERα knockdown decreases Orai3 mRNA (by ∼63%) and protein (by ∼44%) with no effect on Orai1. ERα knockdown decreases Orai3-mediated SOCE (by ∼43%) and the corresponding Ca(2+) release-activated Ca(2+) (CRAC) current (by ∼42%) in ERα(+) MCF7 cells. The abrogation of SOCE in MCF7 cells on ERα knockdown can be rescued by ectopic expression of Orai3. ERα activation increased Orai3 expression and SOCE in MCF7 cells. Epidermal growth factor (EGF) and thrombin stimulate Ca(2+) influx into MCF7 cells through Orai3. Orai3 knockdown inhibited SOCE-dependent phosphorylation of extracellular signal-regulated kinase (ERK1/2; by ∼44%) and focal adhesion kinase (FAK; by ∼46%) as well as transcriptional activity of nuclear factor for activated T cells (NFAT; by ∼49%). Significantly, Orai3 knockdown selectively decreased anchorage-independent growth (by ∼58%) and Matrigel invasion (by ∼44%) of ERα(+) MCF7 cells with no effect on ERα(-) MDA-MB231 cells. Moreover, Orai3 knockdown inhibited ERα(+) cell tumorigenesis in immunodeficient mice (∼66% reduction in tumor volume). These data establish Orai3 as an ERα-regulated channel and a potential selective therapeutic target for ERα(+) breast cancers.

Emerging roles of Orai3 in pathophysiology

“…73 Ectopic expression of Orai3 in these ER − breast cancer cells results in a smaller thapsigargin-activated SOCE and its potentiation by 2-APB (Fig. 2B), suggesting functional activation of Orai3-mediated SOCE in these cells (potentiation by 2-APB is a characteristic of SOCE mediated by Orai3 [58][59][60][61][62] 25,26 which is potentiated by addition of 30 μM 2-APB to the bath solution (Fig. 2C).…”

“…64 Data from our lab showed that SOCE mediated through Orai3 is required for activation of pro-proliferative and pro-migratory pathways; Orai3 knockdown inhibited NFAT transcriptional activity, as well as Ca 2+ -dependent phosphorylation of ERK1/2 and focal adhesion kinase (FAK). 26 As mentioned above, STIM1 and Orai1 were implicated in mediating breast cancer cell migration in vitro using ER − MBA-MD231 breast cancer cells as well as in mediating metastasis in vivo in immune-compromised mice. 19 Although STIM1 and Orai1 knockdown result in inhibition of ER -breast cancer metastasis in nude mice, their potential use as therapeutic targets is questionable since functional STIM1/Orai1-mediated CRAC pathway is essential for proper immunological responses.…”

“…1). There are reported instances where Orai3 was shown to encode SOCE in a subset of breast cancer cells, expressing the estrogen receptors [23][24][25][26] and in mature effector T cells, 27 but in general SOCE is mediated by Orai1 homomultimeric channels. In fact, in the absence of functional Orai1 channels, ectopically expressed Orai3 can only partially compensate for Orai1 in mediating SOCE in HEK 293 and human fibroblasts.…”

“…However, it is quite intriguing that upon Orai3 knockdown, endogenous Orai1 does not substitute for Orai3 in mediating SOCE and CRAC channel function in ER + MCF7 cells, 23,25,26 suggesting a more complex scenario occurs under native conditions that cannot be explained by changes in STIM1/Orai stoichiometry alone. One possible reason for the inability of native Orai1 to compensate for absence of Orai3 and form a functional CRAC channel in MCF7cells could be the recently reported constitutive binding of Orai1 to the secretory pathway Ca 2+ -ATPase 2(SPCA2) in MCF7 cells.…”

“…An siRNA against Orai3 that achieved ~54% knockdown of Orai3 protein levels affected in vitro invasion of MCF7 cells by ~44% with no effect on the invasive capabilities of ER − MDA-MB231 cells. 26 Greater levels of Orai3 knockdown (~68%) were achieved using lentivirusencoding shRNA against Orai3. Stable knockdown of Orai3 was achieved in MCF7 cells using lentiviral infection, and these cells were subsequently injected into the mammary fat pads of severe combined immunodeficient (SCID) mice.…”