ORAI1-Mediated Calcium Influx in Lactation and in Breast Cancer

The characterization of ORAI in breast cell lines. A, the expression of ORAI1, ORAI2, and ORAI3 mRNA in breast cell lines; nonmalignant 184A1 and 184B5 and malignant BT-483, MCF-7, ZR-75–1, MDA-MB-321, and T-47D. Data are fold change, normalized to 18S rRNA, and relative to 184A1. Quantitation was based on 2 independent real time RT-PCRs carried out in triplicate. B, inhibition of ORAI1 in MCF-7 and MDA-MB-231 breast cancer cell lines using siRNA compared with nontargeting siRNA (NT siRNA) and mock transfected cells (Mock). The data are mean ± SEM (N = 6) and are from 2 independent experiments. C and D, the effect of ORAI1 inhibition on store-operated calcium influx in MCF-7 (C) and MDA-MB-231 (D) cells. The left panel depicts results typical of 3 independent experiments in each cell line, where relative [Ca²⁺]_I is represented as response over baseline. Data are from individual wells. The first arrow corresponds to the addition of the calcium chelator BAPTA (500 μmol/L), the second arrow (where relevant) refers to the addition of the SERCA inhibitor CPA (10 μmol/L) to deplete calcium stores, and the third arrow to the addition of extracellular Ca²⁺ (2 mmol/L) to assess store-operated calcium influx. The right panels represent pooled data for the peak ratio (mean ± SEM; N = 12) consisting of the maximal response over baseline value during the Ca²⁺ influx transient divided by the maximal value obtained during the CPA-induced Ca²⁺ transient. This more directly assesses store-operated calcium influx through correction of alterations in the degree of calcium store release. *P < 0.05 compared with NT siRNA using a 1-way ANOVA and a Dunnett's multiple comparison post test. E, ORAI1 levels are higher in the basal breast cancer subtype (P = 0.00063, Student's t- test) Oncomine [Version 3.6, Compendia Bioscience, Ann Arbor, MI; (55)].

The effect of ORAI1 inhibition in breast cancer cells. A, ORAI1 was inhibited using ORAI1 specific siRNA, and the effect on cell growth in MDA-MB-231 and MCF-7 cells was measured using an MTS assay and compared with nontargeting siRNA (NT siRNA) and mock transfected cells (Mock). Data are percentage of NT siRNA, mean ± SEM (N = 12) from 3 independent experiments. *P < 0.001 using a 1-way ANOVA with a Bonferroni multiple comparison posttest to compare NT siRNA with ORAI1 siRNA for each cell line. B, ORAI1 was inhibited using the nonspecific calcium channel antagonist 2-APB and cell growth was compared with control using an MTS assay. Data are mean ± SEM (N = 8) from 2 independent experiments. *P < 0.001 compared with control using a 2-tailed unpaired t test. C, remodeling of the calcium influx signal in MDA-MB-231 breast cancer cells in the presence of the PAR-2 receptor activator trypsin and ORAI1 inhibition. Results are typical of 3 independent experiments, where relative [Ca²⁺]_I is represented as response over baseline. Data are from individual wells. The first arrow corresponds to the addition of the calcium chelator BAPTA (500 μmol/L), the second arrow (where relevant) to the addition of the trypsin (100 nmol/L), and the third arrow to the addition of extracellular Ca²⁺ (2 mmol/L) to assess PAR-2-mediated calcium influx. D, pooled data for the peak ratio (mean ± SEM; N = 10–12), which consists of the maximal response over baseline value during the Ca²⁺ influx transient divided by the maximal value obtained during the trypsin-induced Ca²⁺ transient. *P < 0.001 using a 1-way ANOVA with a Bonferroni multiple comparison posttest to compare NT siRNA with ORAI1 siRNA. E, ORAI1 expression was inhibited in MDA-MB-231 cells using ORAI1 siRNA and the effect on the percentage of cells in S phase was assessed. Data are from 3 independent experiments carried out in triplicate (mean ±SEM).

Stim isoform expression in the mouse mammary gland during development and the effect of Stim2 inhibition on basal calcium levels in mammary epithelial cells. For Stim 1 (A) and Stim 2 (B) values are normalized to 18S rRNA and are relative to the nulliparous sample. Each dot represents mean values from 1 mouse derived from 2 independent real time RT-PCRs, carried out in triplicate. C, Stim2 siRNA reduced basal cytosolic calcium levels compared with the nontargeting control. Data are mean ± SEM from 4 independent experiments carried out in quadruplicate. *P < 0.05 compared with nulliparous gland using a 1-way ANOVA with Tukey posttest (Panel A and B) or using a paired t test (Panel C).

STIM1 and STIM2 mRNA levels in nonmalignant breast cell lines and breast cancer cell lines. A, STIM1 and STIM2 are not overexpressed in breast cancer cell lines relative to the 184A1 and 184B5 nonmalignant breast cell lines. Data are fold change normalized to 18S rRNA and relative to the 184A1 cell line and are depicted as means derived from 2 independent real time RT-PCRs carried out in triplicate. B, the ratio of STIM1/STIM2 was calculated using ΔCt values normalized to 18S rRNA, then calculating STIM1 expression relative to that of STIM2 for each cell line.