Blockage of LMP1-modulated store-operated Ca2+ entry reduces metastatic potential in nasopharyngeal carcinoma cell

Cancer Letters 360 (2015) 234–244

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Cancer Letters j o u r n a l h o m e p a g e : w w w. e l s e v i e r. c o m / l o c a t e / c a n l e t

Original Articles

Blockage of LMP1-modulated store-operated Ca2+ entry reduces metastatic potential in nasopharyngeal carcinoma cell Jiazhang Wei a,b,*, Jinyan Zhang c,d, Yongfeng Si b, Masamitsu Kanada e, Zhe Zhang f, Susumu Terakawa c, Hiroshi Watanabe a a

Department of Clinical Pharmacology and Therapeutics, Hamamatsu University School of Medicine, 1-20-1 Handayama, Hamamatsu 431-3192, Japan Department of Otolaryngology-Head and Neck Oncology, The People’s Hospital of Guangxi Zhuang Autonomous Region, 6 Taoyuan Road, Nanning 530021, China c Medical Photonics Research Center, Hamamatsu University School of Medicine, 1-20-1 Handayama, Hamamatsu 431-3192, Japan d Department of Chemotherapy, Affiliated Cancer Hospital of Guangxi Medical University, 71 Hedi Road, Nanning 530021, China e Department of Pediatrics, Stanford University School of Medicine, Clark Center E150, 318 Campus Drive, Stanford, CA 94305, USA f Department of Otolaryngology-Head & Neck Surgery, First Affiliated Hospital of Guangxi Medical University, Nanning 530021, China b

A R T I C L E

I N F O

Article history: Received 11 November 2014 Received in revised form 11 February 2015 Accepted 12 February 2015 Keywords: Latent membrane protein 1 Store-operated Ca2+ entry Angiogenesis Metastasis

A B S T R A C T

Epstein–Barr virus (EBV)-encoded latent membrane proteins (LMPs) expedite progression of EBVrelevant cancers. Of the full set of LMPs, latent membrane protein 1 (LMP1) was identified to uniquely augment store-operated Ca2+ entry (SOCE). Previously, we reported that the suppression of SOCE exhibited inhibitory effects on cell migration and the extravasation from vasculature in EBV-negative nasopharyngeal carcinoma (NPC) cells. In this follow-up study, we aimed to expand our understanding of the modulation of SOCE by LMP1 and test the possibility that blockage of LMP1-modulated SOCE affects the LMP1-promoted metastatic potential. Here we showed that suppressions of the LMP1-boosted SOCE blunted the LMP1-promoted cell migration, VEGF-mediated angiogenesis and permeabilization in vitro. Blockage of SOCE inhibited vasculature-invasion of circulating cells and distant metastatic colonization in vivo. Notably, utilizing VEGFR2-EGFP-tag zebrafish we revealed that the LMP1-expressing cells arrested in a small-caliber vessel mobilized surrounding endothelial cells to facilitate vasculatureinvasion. Thus, the LMP1-boosted SOCE promotes metastatic potential of NPC cells by solidifying their collaborations with the nearby non-cancer cells through the manipulation of oncogenic Ca2+ signaling. Our study highlights the advantage of using both conventional mammal and transgenic zebrafish for developing a novel therapeutic strategy targeting the multiple steps of invasion-metastasis cascade. © 2015 Elsevier Ireland Ltd. All rights reserved.

Introduction Latent membrane proteins (LMPs) encoded by Epstein–Barr virus (EBV) has been established as a major pathogenetic cause for the development of EBV-related human malignances [1]. As a member of LMPs family, latent membrane protein 1 (LMP1) functions as a key oncoprotein in EBV-associated lymphomas and carcinomas, including Burkitt’s and Hodgkin’s lymphomas, post-transplant lymphoproliferative disease, some of NK/T-cell lymphomas, a part of gastric carcinoma, and nasopharyngeal carcinoma (NPC) [2,3]. LMP1 empowers the EBV-infected cells with diverse malignant properties and thereby contributing to the highly metastatic nature of

Abbreviations: EBV, Epstein–Barr virus; LMP1, latent membrane protein 1; SOCE, store-operated Ca2+ entry; NPC, nasopharyngeal carcinoma; EGF, epidermal growth factor; VEGF, vascular endothelial growth factor. * Corresponding author. Tel.: +86 0771 2186318; fax: +86 0771 2186756. E-mail address: [email protected] (J. Wei). http://dx.doi.org/10.1016/j.canlet.2015.02.032 0304-3835/© 2015 Elsevier Ireland Ltd. All rights reserved.

NPC [4–7]. However, the principle that underlies the progressive evolution from initiatory ectopic LMP1 expression into the invasive cellbiological behaviors in EBV-infected host cells remains unclear. The “tumor microenvironment” constituted by cancer cells and the neighboring multiple cell types, including immune inflammatory cells, endothelial cells, fibroblasts, pericytes and other stromal cells, breeds the progressions of human cancers [8]. To connect with the surrounding non-cancer cells, cancer cells must sense extracellular signals and transmit the signals into cytoplasm. The flow of calcium ions (Ca2+) is a unique membrane-permeable messenger that mediates the signal transmission from the external space into the cytoplasm. Store-operated Ca2+ entry (SOCE) is a pattern of evoking Ca2+ influx in non-excitable cells in response to external chemical and mechanical transmitters [9,10]. It was demonstrated that SOCE was implicated in the growth, angiogenesis and metastasis in cervical and breast cancers [11,12]. Previously, we reported that the inhibition of SOCE suppressed epidermal growth factor (EGF)-stimulated cell migration and the extravasation from vasculature in EBV-negative NPC cells [13]. However, the participation

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of SOCE in EBV-derived malignant profiles was not elucidated in this preliminary study. More importantly, of the full set of the EBVencoded latency genes, LMP1 was identified to interfere in cytosolic Ca2+ homeostasis by enhancing SOCE in B lymphoma cells [14]. Even so, whether LMP1 promotes the invasiveness and metastatic potential in EBV-associated cancer cells through the modulation of SOCE is still unknown. The present study aimed to advance our understanding of the role of SOCE in the LMP1-driven malignant profiles. The exogenous EGF-stimulated cell migration was investigated, which represents the reactiveness in response to the extracellular signal derived from surrounding non-cancer cells. The vascular endothelial growth factor (VEGF)-mediated angiogenesis and permeabilization of endothelium were also studied, which represent the cancer cell-initiated mobilization of the nearby endothelial cells. Alternately using mice and transgenic zebrafish models, we further tested the outcomes of the tumor-associated angiogenic status in primary site, the vasculature-invasion of circulating disseminated cells and the consequential metastatic colonization in vivo upon the blockage of LMP1-modulated SOCE, respectively. Materials and methods Cell culture Human nasopharyngeal carcinoma (NPC) cell lines CNE1 (well-differentiated) and HNE2 (poorly-differentiated) stably expressing EBV-encoded LMP1 were provided by Xiangya Central Experiment Laboratory (Changsha, China), which were established by the introduction of full-length LMP1 cDNA into LMP1-negative CNE1 and HNE2 wild cells, respectively [15–21]. The LMP1 expression was confirmed by western blotting (anti-EBV LMP1 antibody, Abcam Corp., Cambridge, UK). The CNE1 and HNE2 cells transfected with pSG5 empty vector served as the mock control for CNE1-LMP1 and HNE2-LMP1, respectively. The cells were cultured in Dulbecco’s Modified Eagle’s Medium/Nutrient Mixture F-12 supplemented with 5% fetal bovine serum (FBS), penicillin at 100 U/mL and streptomycin at 100 μg/mL (Invitrogen Corp., Carlsbad, CA, USA). Human umbilical vein endothelial cells (HUVECs; ATCC, CRL1730™) were purchased from American Type Culture Collection (ATCC; Manassas, USA), and cultured in Ham’s F-12K Medium (Invitrogen Corp.) supplemented with 10% FBS, heparin at 0.1 mg/mL and the antibiotics. Measurement of cytosolic Ca2+ concentration ([Ca2+]cyto) The cells were preincubated in serum-free medium for 24 h before the measurement of cytosolic Ca2+ to avoid any undesired stimuli to induce Ca2+ responses, and the Ca2+ concentration in the medium was adjusted to 1.8 mmol/L. Cytosolic Ca2+ concentration ([Ca2+]cyto) was measured in individual NPC cells by using an fluorescent Ca2+ indicator, acetoxymethyl ester of fura-2 (fura-2/AM) (Dojindo Molecular Technologies, Inc., Kumamoto, Japan), as our previous description [13,22,23]. RNA interference and western-blotting To achieve a reduced expression of ORAI1, the RNA interference against ORAI1 was performed using a siRNA product (MISSION, Sigma-Aldrich Inc., St Louis, MO, USA), as described in our previous study [13]. For long-term observations in nude/ SCID mice, pre-designed ORAI1-shRNA pLKO.1 plasmid DNA (Sigma-Aldrich Inc.) was transfected into CNE1-LMP1 cells with Lipofectamine 2000 (Invitrogen Corp.) and selected with puromycin (Sigma-Aldrich Inc.) according to the manufacturer’s instructions. The cells transfected with negative control-siRNA or pLKO.1 empty vector plasmid DNA were used as the controls. The reduced ORAI1 expression in siRNA or shRNA-transfected cells was confirmed by western blotting as described previously [13]. α-Tubulin expression was monitored as an in-house control (anti-alpha Tubulin antibody, Abcam Corp.). Cell migration assays Wound healing and Boyden chamber cell migration assays were employed to elucidate the EGF-stimulated cell migration in vitro. To avoid the cellular debris or cytoplasmic contents from inducing any undefined effect on cell migration or Ca2+ responses, which generally results from the scrapping cell monolayer, CultureInserts (Ibidi GmbH, Martinsried, Germany) were utilized. In brief, cells were seeded into the two reservoirs of Culture-Inserts placed on the bottom of a 35 mm dish, which allowed the cells in each separated culture reservoir to grow into a confluent monolayer. A cell-free gap (500 μm in width) was formed after the removal of the Culture-Inserts. Boyden chamber cell migration assays were performed as in our previous study [13].

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VEGF-mediated angiogenesis assay in vitro A total of 5 × 104 NPC cells suspended in medium were seeded into each well of 6-well plate and were routinely cultured until achieving approximately 80% confluent monolayer. The cells were incubated with serum-free medium for 12 h. The medium containing EGF at 50 ng/mL and 0.5% serum was applied to induce VEGF release in the serum-starved NPC cells for 12 h. The amount of VEGF in the conditional medium was determined by an ELISA kit according to the manufacturer’s protocol (VEGF Human ELISA Kit, Invitrogen Corp.). The conditioned medium was harvested for subsequent tube-formation angiogenesis assay as described in previous studies [24,25]. The total tube length in each well was measured to evaluate the angiogenesis using WimTube Image Analysis (Ibidi GmbH).

Endothelial permeability assay HUVECs were seeded onto the upper chambers of BD Falcon™ Cell Culture Inserts (3.0 μm pore size, high pore density, BD Biosciences, Franklin Lakes, NJ, USA) and allowed to achieve complete confluence occluding the polyethylene terephthalate permeable membranes. The endothelial monolayers were incubated in serum-free medium for 24 h before the test of permeability to avoid any undesired stimulation. The NPC cell-conditioned medium containing VEGF was added into both upper and bottom chambers. FITC-Dextran (MW = 70 kDa, final concentration at 1 mg/ mL) was simultaneously applied into the endothelial monolayer-coated upper chambers. The diffusion of FITC-dextran through the cell monolayer into bottom chamber was determined to evaluate the endothelial permeability. The medium in bottom chamber was sampled at indicated time and the fluorescent density was measured at Ex/Em = 480/530 nm.

Nude mouse xenograft All the in vivo experiments performed in the present study conformed to the Guide for the Care and Use of Laboratory Animals published by the US National Institutes of Health (NIH publication, eighth edition) [26]. All experiments were carried out in accordance with the regulations of the Animal Research Committee of Hamamatsu University School of Medicine. A total of 5 × 106 CNE1-LMP1 cells transfected with ORAI1-shRNA or control-shRNA were resuspended in sterile PBS and subcutaneously inoculated into the left flank of each nude mouse (Japan SLC, Inc. Hamamatsu, Japan). Six mice were assigned into each group. Ten days after the inoculations the palpable tumors were observed. The tumor dimensions were measured every five days using a vernier caliper and the tumor volumes were calculated according to the formula: V = (W2 × L)/2, where W was width and L was length. The number of the subcutaneous tumor-associated vessels generated from each branch on xenograft surface was counted for quantitative analysis of angiogenesis status.

Zebrafish hematogenous metastasis model A transgenic strain of zebrafish expressing enhanced green fluorescent protein (EGFP) under the flk1 (VEGFR2) promoter (flk1: EGFP) was purchased from Zebrafish International Resource Center (Univ. Oregon, Eugene, USA) [27,28]. The zebrafish hematogenous metastasis model was established for investigating the intravascular metastasis of NPC cells in our previous work [13]. Suspended siRNA-transfected CNE1LMP1 cells stained with a long-term traceable fluorescent dye (SNARF-1; Invitrogen Corp.) were microinjected into the common cardinal vein as described previously [13]. The blood-transmitted CNE1-LMP1 cells arrested in a small-caliber vessel was observed and the fluorescence images were captured under a confocal microscope (FV1000, Olympus Corp., Tokyo, Japan).

Lung metastatic colonization in SCID mice A total of 5 × 106 CNE1-LMP1 cells transfected with ORAI1-shRNA or controlshRNA in 0.2 mL sterile PBS were injected into the lateral tail vein of Severe Combined Immunodeficiency (SCID) mice (CLEA Japan, Inc., Tokyo, Japan), as described previously [9]. Six mice were assigned into each group. On the seventh day postinjection, the mice were anesthetized and perfused with PBS containing 4% paraformaldehyde, then the mice were sacrificed and the lungs were isolated. The lung tissues were subjected to paraffin-embedded section and followed by routinely staining with hematoxylin and eosin (H&E). The metastatic nodule whose longest diameter ≥100 μm was defined as colonization, and the metastasis whose longest diameter <100 μm was defined as micrometastasis.

Statistical analysis All graphical values were represented as mean ± standard error (s.e.m.). Unpaired Student’s t-test or Fisher’s exact test were used for statistical analyses. Statistical significance was assumed at P < 0.05 and denoted with asterisk.

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Results LMP1 amplifies EGF-stimulated SOCE while promoting cell migration We first studied the influence of LMP1 expression on EGFinduced Ca2+ signaling. The instantaneous Ca2+ responses stimulated by EGF (100 ng/mL) were examined in an immortalized nasopharyngeal epithelial cell line NP69, various EBV-negative NPC cell lines and the EBV-positive NPC cell line C666-1. The Ca2+ responses evoked in NP69 cells was significantly weaker than any other NPC cells (Fig. S1). On the other hand, the Ca2+ responses were apparently stronger in C666-1 cells in comparison with the other EBV-negative cells (Fig. S1). The introduction of LMP1 did not affect the resting Ca2+ level in the absence of external stimulation, but the intensity of EGF-evoked Ca2+ responses was significantly amplified in LMP1expessing cells compared with the parental mock-control cells (Fig. 1A). Since this EGF-stimulated Ca2+ responses actually reflected the mobilizations of Ca2+ by two pathways: 1) the membrane receptor (EGFR) activation-induced Ca2+ release from the Ca2+ store (endoplasmic reticulum, ER), and 2) the following Ca2+ influx via SOCE, we next individually examined the effects of LMP1 on these two Ca2+ sources according to the standard complete protocol [29]. LMP1 did not affect the Ca2+ release from ER in the absence of extracellular Ca2+, but significantly raised the peak of the Ca2+ elevation resulting from Ca2+ influx (Fig. 1B). In addition, thapsigargin, a noncompetitive inhibitor of ER Ca2+-ATPase was used to directly deplete the Ca2+ store independently of EGFR activation. Consistently, LMP1 had no effect on ER-released Ca2+, but significantly boosted the subsequently evoked SOCE (Fig. 1C). Moreover, quantitative analysis of western-blot revealed that the expression of ORAI1 (calcium releaseactivated calcium modulator 1), which serves as a pore subunit on the plasma membrane enabling the Ca2+ influx of SOCE, was enhanced in LMP1-expressing cells (Fig. 1D). Therefore, the upregulation of ORAI1 was found in not only LMP1-expressing B lymphoma cells [14], but also the LMP1-expressing NPC cells, which suggests that LMP1 augments SOCE by enhancing ORAI1 expression in EBV-relevant cancer cells regardless of the histological categorization of cellular origin. With the stimulation of EGF (100 ng/mL), the CNE1-LMP1 and HNE2-LMP1 cells migrating at the leading edge were observed to be more invasive than the mock-control cells (Fig. 1F). The disappearance of cell–cell adhesion allowed these leader cells to migrate farther. Some of these cells appeared in an isolated, fibroblast-like shape which was distinct from the epithelial cobblestone-like morphology (Fig. 1F). In contrast, the morphological alteration was observed in neither the parental cell lines without LMP1 (Fig. 1F) nor other EBV-negative NPC cell lines in our previous work [13]. The Boyden chamber and wound healing assays showed that LMP1 significantly promoted EGF-stimulated cell migration (Fig. 1E and F). We also detected ORAI1 expression in the migrating leader CNE1LMP1 and HNE2-LMP1 cells shown in Fig. 1F. The location of ORAI1 expression was consistent with the LMP1-negative cell shown in our previous study [13], confirming that ORAI1 was also constantly expressed while undergoing this pattern of migration (Fig. S2).

(Fig. 2C). The inhibitions of SOCE by either pharmacological blocker or ORAI1-knockdown showed antagonistic effect on EGF-stimulated cell migration (Fig. 2D and E). The blocking of SOCE using 2-APB was almost complete (Fig. 2A), but 2-APB could not eliminate the cell migration. These results should be ascribed to the fact that EGF induces cell migration through other pathways independent of cytosolic Ca2+, such as the EGFR-Src/FAK pathway. Blockage of SOCE suppresses LMP1-promoted angiogenesis and permeabilization LMP1 was reported to promote tumor-associated angiogenesis in NPC [31–33], and the Ca 2+ signaling via SOCE was demonstrated to manipulate VEGF-mediated angiogenesis [11]. Here we examined the release of VEGF by determining the amount of VEGF in the cell-conditioned medium. The spontaneous release of VEGF was relatively low in both the mock-control cells and LMP1expressing cells in the absence of external stimuli (Fig. S3). In contrast, the amount of VEGF was significantly increased upon external stimulation (EGF at 50 ng/mL plus 0.5% serum), and the concentrations of VEGF release from CNE1-LMP1 and HNE2-LMP1 cells were significantly increased in comparison with their parental cells, respectively (Fig. S3). Blockages of SOCE decreased the amount of VEGF release from CNE1-LMP1 and HNE2-LMP1 cells, respectively (Fig. 3A). Furthermore, the medium harvested from the culture of ORAI1-depleted NPC cells exhibited a weaker efficacy to induce endothelial tube formation (Fig. 3B). We also confirmed that the proangiogenic effect was interfered by blocking VEGF signaling in endothelial cell with sunitinib (2.5 μmol/L), an inhibitor for VEGFR2 (Fig. 3B). In addition, we tested the effect of the conditioned medium on the permeability of endothelium in vitro. The diffusion of FITC-dextran through the HUVECs-coated membrane was negligible as compared to the uncoated membrane (Fig. S4). The low permeability for FITC-dextran reflected the function of physiological endothelial barrier. The conditioned medium harvested from the culture of LMP1-expressing cells exhibited permeabilizing effect on the endothelial monolayer and this effect was reduced by the ORAI1-siRNA treatment (Fig. 3C). The tumor-associated angiogenesis caters to the requirements of abundant nutrients and oxygen for the sustained neoplastic growth, as well as the removals of carbon dioxide and metabolites due to the excessively active metabolism [8]. We thus studied tumor growth in xenograft-bearing nude mice. We found that shRNA-mediated depletion of ORAI1 had no inhibitory effect on CNE1-LMP1 cell proliferation in vitro (Fig. 3D and E). However, ORAI1-depletion significantly inhibited the newly generated vessels on the ORAI1-shRNA xenograft surfaces (Fig. 3F and G) and decreased xenograft growth in vivo (Fig. 3H). The typical features of pathologically developed tumor vessels were found in controlshRNA xenografts, such as the distorted and enlarged vessels, deregulated and excessive branching [8], which were decreased in ORAI1-shRNA xenografts (Fig. 3F). Blockage of SOCE disrupts mobilization of neighboring endothelial cell and vasculature-invasion

Blockages of SOCE blunt the LMP1-promoted cell migration To validate the possibility that suppression of LMP1-boosted SOCE can blunt the LMP1-promoted cell migration, we employed two alternative approaches to achieve functional inhibition of SOCE independently of Ca2+ release from ER, one was the blocking of the Ca2+ channel with a pharmacological inhibitor 2-APB [30], and another was a siRNA-mediated knockdown of ORAI1. Effects of 2-APB (20 μmol/L) and ORAI1-knockdown on EGF-evoked ER-released Ca2+ and SOCE were examined, respectively (Fig. 2A and B). The reduced expression in ORAI1-siRNA cells was confirmed by western-blot

Since the difference in tumor size might result in a bias for the analysis of the angiogenesis status, the comparative analysis performed at the single cell level was required to further support the findings in xenografts. On the other hand, the increased release of VEGF potentially alters the pattern of the interaction between NPC cell and endothelial cell. To further clarify these issues, we employed a zebrafish hematogenous model established in our previous work to illustrate the interplay between single circulating LMP1expressing cell and VEGFR2-expressing endothelial cell [13]. The green fluorescent protein (GFP) not only served as a tag for VEGFR2

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Fig. 1. LMP1 boosts the Ca2+ responses via SOCE while promoting EGF-stimulated cell migration. (A) Changes in cytosolic Ca2+ concentration ([Ca2+]cyto) was measured in serum-starved cells. [Ca2+]cyto is shown as the fluorescence ratio (F340/F380) of Ca2+-indicator fura-2. Each trace represents the average data from at least 25 single cells. Histogram in the right panel shows the average peak from the baseline. (B) EGF-stimulated Ca2+ release from ER and the Ca2+ influx via SOCE are shown. Histograms in the right panel show the average peak in each phase. (C) Thapsigargin-induced depletion of ER Ca2+ and the subsequently evoked SOCE. (D) Western blots of LMP1 and ORAI1 expression. α-Tubulin served as the in-house control. The right panel shows the relative expression of ORAI1 quantified by densitometric analyses. (E) Boyden chamber assay. Histogram indicates the average number of transmigrated cells from a random field; n = 6 per group. (F) Wound healing assay for EGF-stimulated cell migration. Representative photographs of migratory cells were captured at 12 h post-administration of EGF. Insets indicate the isolated migratory cells with morphological alteration. The cell migration was quantified by measuring the maximum migration distance in each well at indicated time-point (right panels); n = 5 per group. Data are representative of three independent experiments and presented as mean ± s.e.m. (*P < 0.05, Student’s t-test).

expression in endothelial cell but also marked the lumen of circulatory vasculature (Fig. S5). This model has been utilized to study the VEGF-mediated tumorous angiogenesis in a previous study [34]. The microinjected red fluorescence protein (RFP)-labeled LMPCNE1 cells were carried by the blood flow and then clogged the caudal artery (Fig. 4A). The cluster of CNE1-LMP1 cells was generally arrested in the similar site near the vasculature-corner for approximately six to eight hours (Fig. S5), thus allowing us to observe

how these LMP1-expressing NPC cells interplay with the neighboring endothelial cells. The CNE1-LMP1 cells arrested in a small-caliber vessel actively recruited the surrounding endothelial cells to participate in the vasculature-invasion. Remarkably, the endothelial cells were driven to migrate toward the surface of CNE-LMP1 cells and generated endothelial networks covering the cluster of cells (Fig. 4B, siCtrl-1). The endothelial cells also penetrated into the cell–cell gap of the

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Fig. 2. Inhibitions of SOCE antagonize the LMP1-promoted cell migration. (A) Effects of 2-APB on ER-released Ca2+ and the Ca2+ influx via SOCE are shown. DMSO served as the vehicle control for 2-APB. (B) Effects of ORAI1-siRNA on ER-released Ca2+ and the Ca2+ influx via SOCE. (C) The reduced expression of ORAI1 in siRNA-transfected cells was confirmed by western-blotting. (D) Boyden chamber assay for EGF-stimulated cell migration. The average number of transmigrated cells in a random field is shown; n = 6 per group. (E) Wound healing assay for EGF-stimulated cell migration. Representative photographs of the migratory cells captured at 12 h post-application of EGF are shown. The maximum migration is indicated in each right panel; n = 5 per group. Data are representative of three independent experiments and presented as mean ± s.e.m. (*P < 0.05, Student’s t-test).

CNE-LMP1 cells (Fig. 4B, siCtrl-2). The CNE-LMP1 cell tightly adhering to the endothelium showed a strong tendency to extrude through the weakness of the over-stretched endothelium (Fig. 4B, siCtrl-2). The continuous endothelium of regional vasculature was disrupted and the leakage was eventually formed (Fig. 4B, siCtrl3). ORAI1-depletion in LMP1-CNE1 cell almost abolished the mobilization of endothelial cell (Fig. 4B, siORAI1-1). During the period of the cells arresting in the regional vasculature, the visible endothelial disruption was found in 83.3% (5/6) of six independent observations for control-siRNA cells, but in none for ORAI1-siRNA cells (0%, 0/6). The extravasated cells were occasionally found in the observation of ORAI1-siRNA cells (Fig. 4B, siORAI1-2). But even so, the endothelium in this observation remained intact (Fig. 4B,

siORAI1-2). The cluster of the circulating disseminated cells blocked in a regional vasculature for more than two hours, which stopped the blood flow in the meanwhile, was considered as an embolus. These ORAI1-depleted cells arrested in the regional vasculature for eight hours and had been developed into a severe embolus are shown (Fig. 4B, siORAI1-2). However, no visible disruption of endothelium could be observed. These observations directly revealed that the abundant release of VEGF enabled LMP1-expressing cell to mobilize nearby endothelial cell to reach sufficient cell–cell contact. This pattern of cell–cell contact resulted in disruption of the continuous endothelium, and the leakage of regional endothelium facilitated the extravasation of LMP-expressing cells from a smallcaliber vessel. The knockdown of ORAI1 almost eliminated the

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Fig. 3. Blockage of SOCE suppresses LMP1-promoted angiogenetic and permeabilizing effects. (A) Amount of VEGF in the cell-conditioned medium was determined by ELISA; n = 6 per group. (B) Human umbilical vein endothelial cells (HUVECs) were incubated with the indicated cell-conditioned medium. DMSO served as the vehicle control for sunitinib. Representative photographs of HUVECs-tube formation were captured at 6 h post-cell seeding; bar = 100 μm. The tube formation was quantitatively analyzed by measuring the tube length per standard area in each well (bottom panel); n = 6 per group. (C) Permeability of HUVECs was tested during the incubations with the indicated conditional medium. The diffusion of FITC-dextran through endothelial cell monolayer was measured at indicated time by measuring the fluorescent density of FITCdextran in bottom chambers; n = 5 per group. Permeability of endothelial monolayer was quantified as the average diffusion rate in 90 minutes. (D) The reduction of ORAI1 expression in shRNA-transfected cells was confirmed by western-blot. (E) Effect of ORAI1-depletion on cell growth in vitro. Cell proliferation was evaluated by determining the cell number in each well of six-well plate using an automated cell counter, n = 6 per group. (F) Representative photographs of xenograft-bearing mice were captured on the 35th day post-inoculation. The subcutaneous newly generated tumor-associated vessels are shown. (G) The average number of tumor-associated vessels generated from each branch on xenograft surface was counted. (H) Tumor volumes were measured every five days from the tenth day post-inoculation; n = 6 per group. Data are representative of three independent experiments (A, B, C, D, E) and presented as mean ± s.e.m. (*P < 0.05, Student’s t-test).

mobilization of surrounding endothelial cells, and thus inhibited the extravasation of CNE1-LMP1 cells by preventing the formation of endothelium leakage. Blockage of SOCE inhibits distant metastatic colonization The capability of cancer cell to migrate is strictly required for the multiple steps of invasion-metastasis cascade, such as the local invasion, the intravasation into lumen of vessels, and the extravasation from the vasculature. The capability to induce angiogenesis, on the other hand, is not only required for the generation of new

vessels around primary tumor nests, but also crucial for disseminated cancer cell to induce neovascularization and form metastatic node in a remote site [35]. Based on the findings shown above, we hypothesized that blockage of SOCE could suppress the metastasis of the circulating LMP1-expressing NPC cell to distant sites. To verify this, we observed the distant metastatic colonization in a lungmetastasis SCID mice model since lung generally is the first site where the circulating disseminated tumor cells create distant metastasis. The state of the lung metastasis in each section was classified as 1) colonization, with or without micrometastasis; 2) only micrometastasis; or 3) none of either one. Of all the lung tissues

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Fig. 4. Blockage of SOCE disrupts mobilization of endothelial cell and vasculature-invasion in zebrafishes. (A) The microinjected CNE1-LMP1 cells were transported along with the blood flow in circulatory vasculature in a VEGFR2-EGFP-tag zebrafish at 2 h post-injection. The RFP-labeled CNE1-LMP1 cells were captured before their arrival to the arresting site. (B) Visualization of the interplay of circulating CNE1-LMP1 cells and nearby endothelial cells. The extended photographs show the clusters of CNE-LMP1 cells that were arrested in the similar site. The anatomical structures of regional vasculature and the pathological alterations of endothelium are illustrated in the rightmost panels. Arrows denote the extravasating cells (white), the extravasated cells (yellow), the embolus-forming cells (blue) and the direction of blood flow (black). Results are representative of six independent observations in each group. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

in control group, the metastatic colonization was found in 83.3% (10/ 12) of the histological lung sections, but only detected in 25.0% (3/ 12) of those in ORAI1-depleted group (Fig. 5A, C and Fig. S6). In these sections with distinct metastatic colonization, the number of metastatic nodules in ORAI1-depleted group was significantly less than that in the control group (Fig. 5B). The micrometastases were found in 8.3% (1/12) and 33.3% (4/12) of the cases in control group and ORAI1-depleted group, respectively (Fig. 5A, C and Fig. S6). Thus, the reduced incidence and severity of metastatic colonization in ORAI1depleted group indicate that the blockage of SOCE in circulating disseminated CNE1-LMP1 cell inhibits the formation of distant metastases (Fig. 5B and C). Discussion Ca2+ signaling controls almost every aspect of cellular functions and enables cells to adapt to external environmental changes

[36]. The cytosolic Ca2+ responses elicited by extracellular molecules like receptor agonists represent the initial signal transduction from the exterior into the cytoplasm. Store-operated Ca2+ entry (SOCE) is an entry of extracellular Ca2+ following the depletion of intracellular Ca2+ stores (endoplasmic reticulum, ER) [9,10]. The Ca2+ release from ER is initiated by the binding of extracellular ligands to the membrane receptors. Since the induction of EGFR by LMP1 has been well characterized in various EBV-relevant tumor cells, including NPC [37–40], it was a possibility that the enhanced EGFstimulated Ca2+ responses resulted from the LMP1-affected EGFR. However, our results showed that LMP1 did not affect the ERreleased Ca2+ linking to the activation of EGFR (Fig. 1B), which imply that the amplified Ca2+ responses should not be ascribed to the LMP1-affected EGFR. Moreover, the SOCE activated by depletion of ER Ca2+ store utilizing an inhibitor of ER Ca2+-ATPase (thapsigargin), instead of EGF, was also augmented in LMP1-expressing cells (Fig. 1C), thus confirming that LMP1 boosts SOCE independently of

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Fig. 5. Blockage of SOCE inhibits distant metastatic colonization. (A) The representative lung sections with histological staining from two separated SCID mice in each group are shown. Equally amplified illustrations (4-folds) of metastatic colonization (big square box) or micrometastasis (small square box) are shown; bar = 100 μm. (B) The number of metastatic nodules per section. The comparison included the sections with detectable metastatic colonization; n = 10 in control group and n = 3 in ORAI1-depleted group. Results are represented as mean ± s.e.m. (*P < 0.05, Student’s t-test). (C) The outcomes of the vein-injection of CNE1-LMP1 cells in control group and ORAI1-depleted group are summarized. (*P < 0.05, Fisher’s exact test).

the upstream pathway, such as the activation of membrane receptor or the depletion of ER Ca2+ content. Cytosolic Ca2+ is a ubiquitous regulator of cell migration. Remodeling of Ca 2+ signaling allows cancer cells to acquire the capabilities to migrate and metastasize [41], and the aberrant reform of SOCE has been proven to correlate with the malignant behaviors of EBV-irrelevant cancer cells [42,43]. LMP1 promotes NPC cell migration by manipulating the epithelial–mesenchyme-transition (EMT) [44–46]. The reductions of cellular junction molecules such as plakoglobin and E-cadherin result in the loss of cell–cell adhesion in LMP1-expressing NPC cells [44–46]. Ca2+ signaling is strictly required for EGF-induced EMT in human breast cancer cells [47]. In this study, we showed that the LMP1-boosted SOCE was responsible for the promotion of EGF-stimulated cell migration. However, the data presented in our study are insufficient to testify the involvement of SOCE in the LMP1-promoted EMT. The participation of LMP1-modulated SOCE in the crucial molecular events during EMT, such as the switch from E- to N-cadherin, still remains to be further clarified. In addition, the enhanced EGF-induced cell migration might not only be attributable to the LMP1-boosted SOCE, but also the LMP1-functionalized EGFR through other parallel pathways [37–40]. Ca2+ signaling regulates not only cell motility but also cell exocytosis [36]. In particular, some of the horizontal transfers of signal molecules derived from tumor cells are Ca2+-dependent and crucial for cancer progression [48]. Tumor-released VEGF serves as a mediator for the extracellular communication between cancer cell and endothelial cell [48]. Cancer cells utilize VEGF to disrupt endothelial barrier function [49,50]. The breakdown of endothelial barrier facilitates the infiltration of local-invaded cancer cell into

circulation, as well as the subsequent extravasation in distant sites. Enhancement of SOCE was demonstrated to promote angiogenesis by increasing VEGF secretion in cervical cancer cell [11]. The enhanced angiogenesis and endothelial permeability mediated by tumor cell-released VEGF potentially increase the risk of hematogenous metastasis. Herein we verified that blockage of LMP1boosted SOCE reduced the NPC cell-released VEGF, and accordingly suppressed the angiogenesis and stabilized the endothelial barrier function in vitro (Fig. 3A, B and C). It should be noted that, the capillaries generated on the xenograft surface might not fully reflect the tumorous angiogenesis (Fig. 3F and G), and the contribution of LMP1-boosted SOCE to angiogenesis is still needed to be further confirmed by determining the blood vessel makers inside the tumor tissue. Interestingly, using a VEGFR2-EGFP-tag zebrafish hematogenous metastasis model, we found that the circulating CNE1LMP1 cells arrested in a small-caliber vessel were capable of recruiting endothelial cells within a short period (6 h). The CNE1LMP1 cell-initiated mobilization of endothelial cells facilitated the vasculature-invasion event. This interplay between circulating cancer cell and endothelial cell was eliminated upon the blockage of SOCE in CNE1-LMP1 cell. Taking together, the blockage of LMP1-boosted SOCE could reduce the VEGF-mediated malignant actions such as the angiogenesis, permeabilization of endothelium and vasculature-invasion. In the present study, we provide pieces of evidence showing that EBV-encoded LMP1 promotes cell migration/invasion and angiogenesis by boosting SOCE. All these properties successively contribute to the endpoint event of human malignances: the distant metastasis (Fig. 6). The distant metastatic colonization shown in our study

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Fig. 6. Blockage of SOCE intervenes in multiple steps of invasion-metastasis cascade. Blockage of LMP1-boosted SOCE exhibited inhibitory effects on multiple steps of hematogenous metastasis.

(Fig. 5A) reflected not only the capability of cancer cell to metastasize to distant sites, but also the adaptation to a foreign environment. We confirmed that blockage of SOCE suppressed CNE1LMP1 cell-formed lung metastatic colonization. The decrease in metastatic colonization might be attributable to the reduced capability of the ORAI1-depleted cell to induce angiogenesis (Fig. 5B and C), which is strictly required for the metastatic formation [35]. However, it has to be noted that other undefined stimuli might also promote the angiogenesis/metastasis in LMP1-expressing NPC cell through the amplified SOCE, and the reductions of angiogenesis and metastatic colonization shown in the in vivo studies actually reflected a total outcome of the attenuations of all these potential proangiogenesis/metastasis stimulations, but not that radiated from any single source. Within the tumor microenvironment, cancer cells constantly receive external stimuli (such as the exogenous growth factors supplied by stromal cells) and produce/release signal molecules (such as VEGF) to recruit the nearby non-cancer cells as needed. In this study, we showed that the boosted SOCE in LMP1-expressing cell enhanced not only the EGF-stimulated cell migration, but also the VEGF-mediated mobilization of endothelial cell. These findings suggest that the LMP1-modulated SOCE promotes the malignant capabilities by solidifying the interactions between the NPC cell and the nearby non-cancer cells, which eventually contributes to the assembling and programming of a tumorous aggressive complex [8]. Dellis et al.’s report strongly suggest that LMP1 may not be the only EBV-encoded protein that can affect Ca2+ signaling in EBVinfected cells [14]. Thus, it is a possibility that the LMP1-effect on cytosolic Ca2+ could be superimposed, or even interpreted in the presence of other EBV-protein. To date, the comprehensive understanding of the modifications of Ca2+ signaling in EBV-infected cells is still

lacking, mainly due to the unknown effects of various EBV-proteins on cytosolic Ca2+. Here we established the role of LMP1 in modification of Ca2+ signaling by introducing LMP1 into EBV-negative NPC cells. However, to fully illustrate the remodeling of cytosolic Ca2+ in EBV-infected NPC cells, the characterization of other EBVproteins, such as LMP2A/B, is still required. Furthermore, the role of each EBV-protein should be confirmed in EBV-positive NPC cells after the knockdown of these proteins, respectively. The zebrafish model used in our study enables us to catch the key events that occur instantaneously during invasion-metastasis cascade [51]. The dynamic process of extravasation of single NPC cell from vasculature was observed for the first time in this vertebrate model in our previous work [13]. The zebrafish model provides a possibility to uncover the malignant behaviors in not only the invasive cancer cell, but also the non-cancer cells, such as the mobilized endothelial cell in the present study. Combination of zebrafish model and mammalian animal offers a powerful means for us to illustrate a full-view of invasion-metastasis cascade. In the present study, we not only propose a previously unrecognized pathway through which EBV-encoded LMP1 endows NPC cell with aggressive properties, but also provide an experimental paradigm of identifying a novel target for anti-metastatic therapy utilizing both transgenic zebrafishes and conventional mice.

Acknowledgments This work was supported by a special grant in aid for a university research program entitled ‘Anti-tumor Research Promoted by Optical Technology’ from the Ministry of Education, Culture, Sports, Science, and Technology in Japan to Hamamatsu University School of Medicine.

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