Orai3 Constitutes a Native Store-Operated Calcium Entry That Regulates Non Small Cell Lung Adenocarcinoma Cell Proliferation

Orai channels have been associated with cell proliferation, survival and metastasis in several cancers. The present study investigates the expression and the role of Orai3 in cell proliferation in non-small cell lung cancer (NSCLC). We show that Orai3 is over-expressed in cancer tissues as compared to the non-tumoral ones. Furthermore, Orai3 staining is stronger in high grade tumors. Pharmacological inhibition or knockdown of Orai3 significantly reduced store operated calcium entry (SOCE), inhibited cell proliferation and arrested cells of two NSCLC cell lines in G0/G1 phase. These effects were concomitant with a down-regulation of cyclin D1, cyclin E, CDK4 and CDK2 expression. Moreover, Orai3 silencing decreased Akt phosphorylation levels. In conclusion, Orai3 constitutes a native SOCE pathway in NSCLC that controls cell proliferation and cell cycle progression likely via Akt pathway.


Introduction
Calcium is a key messenger that regulates proliferation, apoptosis, migration and invasion, which are the main mechanisms implicated in cancer progression [1,2]. It controls G1 progression, G1/S and G2/M transition phases by regulating expression of several calcium-dependent signaling pathways, such as calmodulin, CaM-Kinase, and calcineurin [3,4,5]. Therefore, identification of the pathways involved in Ca 2+ influx regulating the cell proliferation are in the focus of a number of investigations.
In epithelial cells, store-operated Ca 2+ entry (SOCE) is the main route that drives most Ca 2+ -dependent signaling cascades [6,7,8]. Recent studies have reported that Orai1 and Stim1 regulate storeoperated calcium influx [9,10,11], cell proliferation [12,13] and cell cycle progression [13,14,15]. However, studies of principal components of store-operated Ca 2+ entry in lung cancer cells are limited. Indeed, only three recent studies have shown the involvement of Orai1 and TRPC1 in SOCE in lung cancer A-549 cell line [16,17]. Over-expression of Orai1 or down-regulation of TRPC1 decreases the expression of cyclin D (D1 and D3), arrests cells at G1 phase and inhibits the EGF proliferative effect [16,17]. Furthermore, Inhibition of Orai1 reduces the NFkB activation.
It has been demonstrated recently that Orai3 is a part of a native store-operated Ca 2+ entry pathway in the estrogen receptor (ER) positive breast cancer cells [18]. We have previously reported that down-regulation of Orai3 inhibits cancer cell proliferation, contributes to cell cycle arrest in G1 phase, and increases apoptotic cell death [19]. Interestingly, Orai3 silencing does not affect cell proliferation in the non tumorigenic MCF-10A cells [19]. Given the above we propose that Orai3 may be of crucial significance in the human lung cancer where altered calcium homeostasis could favor cell growth. Therefore, the objectives of our study were to determine Orai3 expression in lung cancer tissue samples and to establish its role in cell proliferation and survival using two NSCLC cell lines representing the most common malignant lung tumor. We demonstrate that Orai3 expression is up-regulated in lung cancer tissues, correlates with high tumor grade, and Orai3mediated Ca 2+ entry is crucial to NSCLC cell proliferation.

Patients: ethical agreement
Ethical approval for this study was granted by the ''Comité Consultatif de Protection des Personnes dans la Recherche Biomédical de Picardie'' (2012/43-Nu ID-RCB: 2012-A01542-41-CANIO-Poumon). The study has been performed on 60 healthy and cancer samples obtained from patients with lung adenocarcinoma received a surgical resection, between 2005 and 2011 at the University Hospital of Amiens. Lung tissue samples were collected from fully informed patients provided written consent prior to clinical procedures. Several histological factors representing important prognostic factors for lung adenocarcinoma were measured, e.g. tumor grade of differentiation (1 = well-, 2 = moderately-, and 3 = poorly differentiated), vascular invasion, and necrosis. The other parameters of the epidemiological study are: population characteristics (age, sex), tumor size, lymph node metastasis (number of lymph nodes examined), tumor, and death.

Immunohistochemistry
Immunohistochemistry was performed on 60 tissue samples of lung adenocarcinoma. Briefly, four-micrometer-thick sections of formalin-fixed and paraffin-embedded tissue samples were sliced from the tissue block. Immunohistochemical staining was performed on a Roche Ultra immunostainer, using antibody directed against Orai3 (rabbit polyclonal, 1:100 dilution, Sigma, Saint Louis, USA). This was followed by the avidin-biotinperoxidase complex technique. Reactions were developed using a chromogenic reaction in DAB (diamino-3,39benzidine tetrahydrochloride) substrate solution (DAB, Sigma Fast). Counterstaining was carried out with hematoxylin solution. This antibody is certified for immunohistochemistry by Sigma Inc. A negative control was performed using the same technique without the primary antibody.
Immunostaining levels for Orai3 were determined by subjective visual scoring of the brown stain. Two operators independently evaluated antigen expression.
Because of a heterogeneous staining of the tumor cells, we chose a score of staining that matched level of intensity (0, 1, 2 or 3) multiplied by proportion of cell staining.

Cell-transfection
Transfection of cells was performed using the nucleofection technology according to the Amaxa Biosystems protocol. Cells were transfected with 4 mg of siRNA directed against Orai1 (59-GCC AUA AGA CUG ACC GAC A -39) or Orai2 (59-GCC ACA ACC GUG AGA UCG A-39) or Orai3 [19], with scrambled siRNA as a control (siGENOME Non-Targeting siRNA, Dharmacon Research Inc., Chicago, IL, USA) in V kit solution and X-013 program (Amaxa Biosystems).

Cell viability and mortality
Cell viability and mortality were assessed by Trypan Blue assay. After transfection with si-Orai3, or with functional non-coding si-CTL as previously described, NCI-H23 and NCI-H460 were grown in 35-mm Petri dishes at a density of 20610 4 cells for 72-h.
Cell viability and mortality was assessed using the standard Malassez cell method [19].

Cell cycle analysis
To measure the cellular DNA content, adherent cells transfected by si-Orai3 or si-CTL for 72-h were collected by trypsinization. Cells were pelleted, resuspended in PBS/EDTA, treated with 20 mg/ml RNase A (Sigma-Aldrich), and stained with 50 mg/ml of Propidium Iodide (Sigma-Aldrich). Samples were analyzed on BD Accuri C6 flow cytometer. The percentage of cells in different phases was calculated using FlowJo software (Tree Star, Inc).

Western blot analysis
Whole-cell lysates were prepared with 1% sodium dodecyl sulphate and a protease inhibitor cocktail (Sigma-Aldrich, France). Proteins were separated by denaturing SDS-PAGE and transferred onto nitrocellulose membranes. The primary antibodies

Apoptosis analysis
To evaluate the percentage of apoptotic cells, we measured cell surface exposure of phosphatidylserines by FITC Annexin V Apoptosis Detection Kit I (BD Biosciences Pharmingen, France). Both detached and adherent cells were collected, washed twice in ice-cold PBS and re-suspended in 16 binding buffer. After staining, the samples were analyzed with BD Accuri C6 flow cytometer.

Ca 2+ measurements
Cells were plated onto glass coverslips after transfection with si-CTL or si-Orai3 or without transfection. The cytosolic calcium concentration was measured using FURA-2 loaded cells. Cells were loaded for 1 h at 37uC with 3 mM of Fura-2-AM prepared in culture medium. Before recording, cells were washed twice with the extracellular dye free solution (NaCl 140 mM, KCl 5 mM, CaCl 2 2 mM, MgCl 2 2 mM, D-glucose 5 mM, and HEPES 10 mM, pH 7.4). The glass coverslip was mounted in a chamber on a Zeiss microscope equipped for fluorescence. Fura-2 fluorescence was excited to 350 and 380 nm using a monochromator (Polychrome IV; TILL Photonics, Planegg, Germany), and fluorescence captured by a Cool SNAPHQ camera (Princeton Instruments, Evry, France) after filtration through a long-pass filter (510 nm). Metafluor software (version 7.1.7.0) was used for acquisition and analysis. All recordings were made at room temperature. The cells were continuously perfused with a saline solution and chemicals were added as indicated. The flow rate of the whole cell chamber perfusion system was set to 1 ml/min with the chamber volume of 500 ml.

Statistical analysis
Data are presented as Mean 6 S.E.M., ''n'' refers to the number of experiments. Statistical analyses were performed using Mann-Whitney or paired t-tests, as appropriate using Sigma-Stat 3.0 (Systat Software, Inc.). When more than two conditions were compared, a Kruskal-Wallis one way ANOVA was employed followed by Dunn's Method post-hoc tests using SigmaStat 3.0 (Systat Software, Inc.). Differences were considered significant at p,0.05.

Results
Orai3 is overexpressed in lung adenocarcinoma and correlated with high tumor grade The expression of Orai3 was analyzed by immunohistochemistry in 60 tumors and adjacent non-tumoral tissue samples obtained from the same patient. A strong staining of Orai3 was observed in 66.7% of tumor samples as compared to the nontumoral samples (40/60, p,0.001). A representative Orai3 staining is shown in Figure 1A. Quantitative analysis of the Orai3 expression revealed that Orai3 staining score significantly increased in tumors (0.5360.06, n = 40) as compared to non tumoral epithelial tissue from the same patient (0.2260.01, n = 20, p,0.001, Fig. 1B).
The Orai3 staining score was elevated in higher tumor grade (grade 3; 0.9260.13; n = 16) as compared to low tumor grades (grade 1-2; 0.6160.04; n = 24, p = 0.032, Fig. 1C-D). We also assessed correlations of the Orai3 expression with clinical parameters (age, sex, tumor size, nodal metastasis, vessel invasion, TNM). No correlations were found between Orai3 expression and clinical parameters (see Table 1). Altogether, these results provide evidence that Orai3 is overexpressed in lung adenocarcinoma and the level of its expression correlates with high tumor grade.

NSCLC cells possess store operated calcium entry (SOCE) and express functional Orai3
We characterized SOCE in two NSCLC cell lines: NCI-H23 and NCI-H460. We used a pharmacological inhibitor, Gd 3+ known to inhibit at low concentration native SOCE in many cell types [20,21]. Intracellular Ca 2+ concentration was monitored by ratiometric imaging in the cells loaded with Fura-2. SOCE was monitored through thapsigargin-stimulated Ca 2+ influx. We first perfused 1 mM thapsigargin in Ca 2+ free solution for 7 min. Then, 5 mM Ca 2+ was added. The perfusion of 5 mM Gd 3+ almost completely suppressed SOCE in both cell lines ( Fig. 2A-B, p,0.001, n = 25 for NCI-H23 and n = 28 for NCI-H460).

Down-regulation of Orai3 reduces cell proliferation and induces cell arrest in G1 phase of the cell cycle
A 72-h treatment with si-Orai3 significantly reduced the cell number (4364.1% and 62.562.6% for NCI-H23 and NCI-H460 cells respectively, p,0.001, Fig. 5A-B). We then examined whether inhibition of Orai3 expression causes NSCLC cell death leading to the reduction in cell number. Down-regulation of Orai3 had no effect on cell death of NCI-H23 or NCI-H460 (Fig. S4 A-B). We have also performed cell counting by Trypan Blue exclusion assay in 48-h after Orai3 silencing and we found a 26% decrease of cell viability in both cell lines without any effect on cell mortality (Fig. S4C-D). These experiments allowed us to conclude that Orai3 plays an important role in NSCLC cell proliferation.
In breast cancer cells, we have previously shown that Orai3 silencing induced cell cycle arrest in the G1 phase [19]. We therefore examined possible alterations in the cell cycle in NSCLC cells with the Orai3-knockdown by flow cytometry. Cells were transfected either by si-Orai3 or siRNA-control for 72-h. Figure 5C-D shows the cell cycle distribution of NCI-H23 and  Fig. 5D). At the same time, a decrease in cell number in S and G2/M phases has been observed in both NCI-H23 and NCI-H460 cells transfected with si-Orai3 ( Fig. 5C-D). These data demonstrate that Orai3 knockdown causes a cell cycle arrest at G0/G1 phase in NSCLC cells.
It has been reported that Orai3 affects cell survival, and inhibition of Orai3 increases apoptosis [19]. We therefore investigated the effect of Orai3 inhibition on apoptosis using Annexin V, Propidium Iodide double-staining by flow cytometry. Orai3 silencing failed to induce apoptosis in both cell lines (Fig. 5E-F).

Orai3 regulates Cyclins and cdk expression
To further clarify the mechanism by which Orai3 knockdown impacts the cell cycle of lung cancer cells, we analyzed the expression of the main cell cycle regulatory proteins by Western blotting. Orai3 silencing decreased the expression of cyclin D1 (49.7621% for NCI-H23 and 79.769.8% for NCI-H460 cells, p,0.05, Fig. 6A-B-C-D), Cdk4 (38.7619% for NCI-H23, 69613% for NCI-H460 cells, p,0.05, Fig. 6A-B-C-D), and Cdk2 (37.4619% for NCI-H23 and 62.6613.7% for NCI-H460 cells, p,0.05, Fig. 6A-B-C-D). Furthermore, down-regulation of Orai3 decreased cyclin E expression by 49.4618.5% in NCI-H460 cells, but was without any effect on cyclin E expression in NCI-H23 cells (Fig. 6A-B-C-D). Altogether, these results indicate the involvement of Orai3 in the cell cycle progression and therefore in cell proliferation.

Discussion
Our results show that NCI-H23 and NCI-H460 cells express Orai1, Orai2, Stim1 and Stim2. SOCE is inhibited by low concentrations of lanthanides (5 mM Gd 3+ ), but neither Orai1, nor Orai2 regulates it in the NSCLC cells. Interestingly, SOCE is increased by 2-APB application and decreased by silencing of Orai3. Importantly, we found that Orai3 contributes to non-small cell lung adenocarcinoma cell proliferation and cell cycle progression likely through Akt pathway. Moreover, Orai3 is overexpressed in tumoral lung tissues compared to normal ones, and correlates with high tumor grade. Since its role in proliferation and its increased expression in NSCLC samples, Orai3 may thus be a potential target for adenocarcinoma lung therapy.
Store-operated Ca 2+ entry mediated by Stim/Orai is involved in the control of several cellular functions, including cell growth and proliferation. Indeed, Orai1, STIM2, but not STIM1 regulate cell cycle progression and proliferation in HEK293 cells [15]. Recently, Motiani et al. [18] have reported the expression of Orai and STIM in five breast cancer cell lines and demonstrated that Orai3 is the major component in the native store-operated channels in ER positive breast cancer cell lines [18]. Furthermore, we have reported that Orai3 knockdown inhibits proliferation of MCF-7 and T-47D human breast cancer cells [19]. Consistent with these studies, the current work provides new evidence that Orai3 is crucial for SOCE and cell proliferation in lung cancer cell lines. In lung adenocarcinoma, predominant studies have been performed on one lung cell line, A-549, where TRPC1 and Orai1 have been reported to contribute to SOCE [16,17]. Thus, there is a crucial need to characterize SOC partners in other NSCLC cell lines. Our data demonstrate that SOCE is sensitive to Gd 3+ in both NCI-H23 and NCI-H460 cells. Moreover, Orai (1-3) and Stim (1)(2) are expressed in these cell lines. Silencing of the Orai1 or Orai2 failed to affect SOCE, while Orai3 knockdown significantly reduced both basal Ca 2+ -fluorescence and SOCE. Our results are in support of Orai3 as the main SOCE pathway in NSCLC cell lines (NCI-H23 and NCI-H460) that participates in the regulation of intracellular Ca 2+ concentration.
Cell proliferation is finely regulated by the progression through three distinctive phases of the cell cycle (G0/G1, S, and G2/M); thus, cell cycle arrest is considered one of most common causes of the inhibition of cell proliferation. We have previously shown in breast cancer cells that Orai3 silencing suppressed cell proliferation, arrested cell cycle in the G0/G1 phase, and this phenomenon is associated with a reduction in CDKs 4/2 and cyclins D1 and E expression. Here, we show that Orai3 controls NSCLC cell growth via regulating G1 to S phase cell cycle transition. Indeed, cyclin D1 and E are essential regulators of G1 and G1/S transition. Our results show that the expression of cyclin D1 and cyclin E, as well as the expression of CDK4/2 were regulated by Orai3. These findings in different types of tumors indicate that Orai3 is likely a key regulator of Ca 2+ -mediated cell cycle progression. In addition, Orai-3 silencing did not induce apoptotic cell death. This result is however different from its anti-apoptotic effect observed in breast cancer [19].
Ca 2+ signaling is required for progression through G1, the G1/ S transition and G2/M in several cell types [25,26,27]. Ca 2+ influx activates Ca 2+ -dependent transcription factors leading to the expression of cell cycle regulatory proteins, such as cyclins, CDKs, and to the inhibition of CDKi expression [28,29,19]. In agreement with these considerations, it has been reported that calcium entry through TRPV6 channel induces a subsequent downstream activation of Nuclear Factor Activated T cell (NFAT) leading to LNCaP prostate cell proliferation [30]. Moreover, in human hepatoma cell line Huh-7, it has been also shown that Ca 2+ entry involving TRPC6, together with STIM1 and Orai1, increases cyclin D1 expression. Furthermore, we have also reported that Orai3 knockdown accumulates the cells in G1 phase and decreases expression of cyclins D1 and E [19]. In this context, we suggest that Ca 2+ entry through Orai3 may control cell cycle progression by regulating expression of cyclins and CDKs in NCI-H23 and NCI-H460 lung cancer cells.
The PI-3K/Akt signaling pathway is central to cell proliferation and survival and is constitutively activated in lung cancer cells [31]. Indeed, blockade of PI-3K/Akt pathway strongly blocks cell cycle along with a decreased expression of cyclins D1 and D3 [17]. Moreover, reducing Ca 2+ entry, via SOCE, inhibits Akt phosphorylation and reduces EGF-induced proliferation in A-549 cells [16]. In the present study, we found that Orai3 silencing suppressed Akt phosphorylation in both cell lines suggesting that the proliferative effect of Orai3 is, in part, attributed to the Akt signaling pathway in lung cancer cells.
Finally, our results clearly show that Orai3 channels are expressed in both normal and tumor lung tissues. However, Orai3 is over-expressed in tissues from 60 patients presenting non small cell lung adenocarcinoma versus non tumoral adjacent tumoral tissues. Moreover, the expression of Orai3 increases in high tumor grade. This result suggests the involvement of Orai3 in NSCLC development.
In conclusion, our study highlights Orai3 as a major regulator of cell cycle progression and therefore growth of NSCLC cells and makes it as an interesting potential bio-marker and/or target for adenocarcinoma lung therapy.