Physiological functions of calcium signaling via Orai1 in cancer

Abstract Intracellular calcium (Ca2+) signaling regulates many cellular functions, including cell proliferation and migration, in both normal cells and cancer cells. Store-operated Ca2+ entry (SOCE) is a major mechanism by which Ca2+ is imported from the extracellular space to the intracellular space, especially in nonexcitable cells. Store-operated Ca2+ entry (SOCE) is also a receptor-regulated Ca2+ entry pathway that maintains Ca2+ homeostasis by sensing reduced Ca2+ levels in the endoplasmic reticulum (ER). In general, the activation of G protein-coupled receptors (GPCRs) or immunoreceptors, such as T-cell, B-cell and Fc receptors, results in the production of inositol 1,4,5-trisphosphate (IP3). IP3 binds to IP3 receptors located in the ER membrane. The, IP3 receptors in the ER membrane trigger a rapid and transient release of Ca2+ from the ER store. The resulting depletion of ER Ca2+ concentrations is sensed by the EF-hand motif of stromal interaction molecule (STIM), i.e., calcium sensor, which then translocates to the plasma membrane (PM). STIM interacts with Orai Ca2+ channel subunits (also known as CRACM1) on the PM, leading to Ca2+ influx from the extracellular space to increase intracellular Ca2+ concentrations. The physiological functions of Orai and STIM have been studied mainly with respect to their roles in the immune system. Based on numerous previous studies, Orai channels (Orai1, Orai2 and Orai3 channels) control Ca2+ release-activated Ca2+ (CRAC) currents and contribute to SOCE currents in other types of cells, including various cancer cells. There are many reports that Orai1 is involved in cell proliferation, migration, metastasis, apoptosis and epithelial–mesenchymal transition (EMT) in various cancers. We previously found that Orai1 plays important roles in cell apoptosis and migration in melanoma. Recently, we reported novel evidence of Orai1 in human oral squamous cell carcinoma (OSCC) cells and human cardiac fibroblasts (HCFs). In this review, we present multiple physiological functions of Orai1 in various cancer cells and cardiac fibroblasts, including our findings

Store-operated calcium entry via ORAI1 regulates doxorubicin-induced apoptosis and prevents cardiotoxicity in cardiac fibroblasts

Despite exhibiting cardiotoxicity, doxorubicin (DOX) is widely used for cancer treatments. Cardiac fibroblasts (CFs) are important in the pathogenesis of heart failure. This necessitates the study of the effect of DOX on CFs. The impairment of calcium (Ca2+) homeostasis is a common mechanism of heart failure. Store-operated Ca2+ entry (SOCE) is a receptor-regulated Ca2⁺ entry pathway that maintains calcium balance by sensing reduced calcium stores in the endoplasmic reticulum. ORAI1, a calcium channel protein and the most important component of SOCE, is highly expressed in human cardiac fibroblasts (HCFs). It is upregulated in CFs from failing ventricles. However, whether ORAI1 in HCFs is increased and/or plays a role in DOX-induced cardiotoxicity remains unknown. In this study, we aimed to elucidate the relationship between ORAI1/SOCE and DOX-induced heart failure. Induction of apoptosis by DOX was characterized in HCFs. Apoptosis and cell cycle analyses were performed by fluorescence-activated cell sorting (FACS). Reactive oxygen species (ROS) production was measured using fluorescence. YM-58483 was used as an ORAI1/SOCE inhibitor. ORAI1-knockdown cells were established by RNA interference. In vivo experiments were performed by intraperitoneally injecting YM-58483 and DOX into mice. We first demonstrated that DOX significantly increased the protein expression level of p53 in HCFs by western blotting. FACS analysis revealed that DOX increased early apoptosis and induced cell cycle arrest in the G2 phase in fibroblasts. DOX also increased ROS production. DOX significantly increased the expression level of ORAI1 in CFs. Both YM-58483 and ORAI1 gene knockdown attenuated DOX-induced apoptosis. Similarly, YM-58483 attenuated cell cycle arrest in the G2 phase, and ORAI1 knockdown attenuated DOX-induced ROS production in HCFs. In the animal experiment, YM-58483 attenuated DOX-induced apoptosis. In HCFs, ORAI1/SOCE regulates p53 expression and plays an important role in DOX-induced cardiotoxicity. ORAI1 may serve as a new target for preventing DOX-induced heart failure

Relationship between DOX, p53, and ORAI1 in CFs.

DOX increased the expression of p53 and induced apoptosis, cell cycle arrest, and ROS production. In addition, DOX increased the expression of ORAI1, not STIM1. Furthermore, the inhibition of ORAI1 negated the DOX-induced expression of p53, suggesting that the DOX-ORAI1-p53 pathway induces cardiotoxicity. (TIF)</p

Western blotting analysis for four groups to evaluate the effects of store-operated Ca²⁺ entry (SOCE) inhibition; CTRL group, DOX group, YM group, and YM+DOX group.

(A) YM-58483 significantly attenuated the DOX-induced upregulation of p53 protein. (B) YM-58483 significantly attenuated the DOX-induced upregulation of p21 protein. (one-way ANOVA followed by Tukey’s test, n = 6, *** p < 0.001, NS: no significant difference).</p

Protocol for western blotting.

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Original data (data shown in Fig 4).

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Western blotting analysis to evaluate the effects of ORAI1 gene knockdown.

(A) Expression of ORAI1 mRNA by qPCR. The expression level of ORAI1 mRNA was reduced in samples with siRNA against ORAI1. In samples with control siRNA, DOX significantly upregulated the expression level of ORAI1 mRNA (two-way ANOVA followed by Bonferroni’s post hoc test, n = 6, *** p p p p p p p p < 0.01, NS: no significant difference).</p

Assessment of DOX-induced cardiotoxicity in mice.

Saline or 40 mg/kg DOX was administered intraperitoneally. In addition, 0.2% DMSO or 10 mg/kg YM-58483 was administered intraperitoneally prior to saline or DOX. (A) TUNEL apoptosis staining in heart sections. Apoptotic nuclei were quantitatively assessed in five random fields. Scale bar, 100 μm. (B) YM-58483 significantly attenuated DOX-induced apoptosis (one-way ANOVA, n = 7, *** p < 0.001, NS: no significant difference).</p

Original data (data shown in Fig 1).

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