Arachidonic acid-induced Ca2+ entry and migration in a neuroendocrine cancer cell line
© The Author(s) 2018
Received: 31 October 2017
Accepted: 24 February 2018
Published: 2 March 2018
Store-operated Ca2+ entry (SOCE) has been implicated in the migration of some cancer cell lines. The canonical SOCE is defined as the Ca2+ entry that occurs in response to near-maximal depletion of Ca2+ within the endoplasmic reticulum. Alternatively, arachidonic acid (AA) has been shown to induce Ca2+ entry in a store-independent manner through Orai1/Orai3 hetero-multimeric channels. However, the role of this AA-induced Ca2+ entry pathway in cancer cell migration has not been adequately assessed.
The present study investigated the involvement of AA-induced Ca2+ entry in migration in BON cells, a model gastro-enteropancreatic neuroendocrine tumor (GEPNET) cell line using pharmacological and gene knockdown methods in combination with live cell fluorescence imaging and standard migration assays.
We showed that both the store-dependent and AA-induced Ca2+ entry modes could be selectively activated and that exogenous administration of AA resulted in Ca2+ entry that was pharmacologically distinct from SOCE. Also, whereas homomeric Orai1-containing channels appeared to largely underlie SOCE, the AA-induced Ca2+ entry channel required the expression of Orai3 as well as Orai1. Moreover, we showed that AA treatment enhanced the migration of BON cells and that this migration could be abrogated by selective inhibition of the AA-induced Ca2+ entry.
Taken together, these data revealed that an alternative Orai3-dependent Ca2+ entry pathway is an important signal for GEPNET cell migration.
In the last decade, Orai1 and stromal interacting molecule-1 (STIM1) were identified as the long sought-after molecular players that are both necessary and sufficient to recapitulate store-operated Ca2+ entry (SOCE) [1–8]. This pathway was shown to be physiologically and pathophysiologically important for a variety of cell types including, lymphocytes and cancer cells [9–11]. Recently, several studies have indicated a role for Orai1 and STIM1 in migration of different types of cancer and non-cancer cells [12–21]. These observations have lead to the consensus that SOCE is important for tumor cell migration . Interestingly, alternative pathways that utilize many of the same molecular constituents of SOCE have been shown to contribute to other less well-characterized modes of Ca2+ entry [23, 24]. However, these alternative pathways have not been adequately investigated in the context of tumor cell migration. For example, the intracellular lipid second messenger, arachidonic acid (AA) and/or its downstream metabolite leukotriene-C4 (LTC4) have been shown to induce a store-independent mode of Ca2+ entry via a plasma-membrane channel that is comprised of both Orai1 and Orai3 [14, 25].
Although some studies indicated that AA or its metabolites such as prostaglandins and leukotrienes might stimulate migration and epithelial to mesenchymal transition (EMT) in some cancer and non-cancer cells [26–29], a role for AA-induced Ca2+ entry has not been examined. Therefore, we ascertained whether SOCE and/or AA-induced Ca2+ entry pathways contributed to cell migration in a tumor-cell line that displayed both pathways.
Previous research from our laboratory had shown that several model cell lines of gastroenteropancreatic neuroendocrine tumors (GEPNETs) expressed mRNA message for Orai homologs and exhibited SOCE . In the current study, we extended these observations to assess whether exogenously administered AA could evoke Ca2+ entry and/or enhancement of cell migration in BON cells, a well-characterized GEPNET cell line.
Using live cell fluorimetric imaging of Ca2+ dynamics in combination with pharmacological treatments, we demonstrated that Ca2+ entry in this cell type could be induced by artificially depleting the ER stores or by exogenous application of AA. We also demonstrated that these modes of Ca2+ entry could be evoked and perturbed selectively. We identified the Orai channels that contributed to these two Ca2+ entry pathways using shRNA-mediated gene knockdown. These studies revealed that expression of Orai1 was required for SOCE and that the AA-induced pathway required both Orai1 and Orai3. Furthermore, we assessed the relative roles of SOCE and AA-induced Ca2+ entry in BON cell migration using a modified Boyden chamber assay. Selective stimulation or perturbation of these Ca2+ entry modes using a variety of pharmacological and molecular tools revealed that under our experimental conditions the AA-induced Ca2+ entry was the dominant Ca2+-signal responsible for BON cell migration. Our results suggest that Ca2+ entry through an Orai3-containing channel is a novel signal for BON cell migration and identifies this pathway as a potential target to limit recurring GEPNET metastasis.
Cyclopiazonic acid (CPA) was purchased from Calbiochem. Arachidonic acid was obtained from MP Biomedicals. Thapsigargin and SK&F 96365 (SKF) were purchased from Tocris Bioscience. Ketoprofen, ethylene glycol tetraacetic acid (EGTA) and 2-aminoethoxydiphenyl borate (2-APB) were obtained from Sigma. Leukotriene C4 (LTC4) and Nordihydroguaiaretic acid (NDGA) were purchased from Cayman chemical.
Cell culture and transfection
BON cells were cultured in flasks with a 1:1 solution of DMEM and F12K supplemented with 10% fetal bovine serum (FBS) and 1% penicillin–streptomycin and maintained at 37 °C in a humidified incubator with 5% CO2. All cell culture reagents were obtained from Life Technologies, unless specifically indicated. For cell transfection, 5 × 106 BON cells were electroporated with plasmid vectors containing shRNAs against Orai1 or Orai3 (OriGene Technologies) using the Amaxa nucleofector II device (Lonza) as per manufacturer’s instruction. All available shRNA constructs supplied by the manufacturer resulted in comparable knockdowns for each subunit, and the data from these experiments were pooled. Transfection efficiency was estimated to be approximately 90% for both constructs. Control cells were transfected with an identical plasmid that contained a scrambled message. Moreover, mock-transfected and untransfected cells were used as additional controls. All plasmids expressed either GFP or RFP and a gene for puromycin resistance. Transfected cells were maintained 2 days in culture medium containing 0.2 μg/ml puromycin dihydrochloride. In addition to western blotting (see below), knockdown of Orai1 and 3 were functionally verified using live cell Ca2+ imaging at different time points and the maximum effects were observed at 48 h after transfection.
Lysates were prepared from BON cells at 48 h post-transfection by extraction of cellular proteins in RIPA buffer (containing 25 mM Tris–HCl, pH 7.6, 150 nM NaCl, 1% Triton X-100, 1% sodium deoxycholate, 0.1% SDS). The cell lysates were concentrated and desalted using the YM-100 Microcon centrifuge filter (Sigma Aldrich). Approximately, 50 μg of concentrated cell lysates were run on 8% Bis–Tris gels under denaturing conditions. Following separation, the protein bands were transferred onto polyvinylidene fluoride (PVDF) membranes for 90 min using the Criterion blotter (Biorad) under semi-wet conditions. The membranes were then incubated for 2 h in a solution of 5% non-fat dry milk in TBS-T buffer (containing in mM, 20 Tris/HCl, 150 NaCl and 0.1% Tween-20) to block non-specific interactions. The membranes were then cut in two and incubated overnight at 4°C with rabbit polyclonal antisera against Orai1 or Orai3 (Prosci). The membranes were washed with TBS-T buffer and probed for 2 h using an HRP-conjugated anti-rabbit secondary antibody (Millipore). Finally, the blots were washed with TBS-T buffer and treated for chemiluminescence visualization using the ECL Western Blotting substrate (Pierce). Detection of protein bands was performed using a LAS 3000 imaging system (Fujifilm). After matching the position of the bands of interests with respect to the molecular mass reference ladder (Invitrogen), the membranes were stripped using Restore solution, (Pierce) and re-probed with HRP-conjugated mouse monoclonal antibody against β-actin (Abcam) and visualized by chemiluminescence. Quantification of band density was achieved using NIH ImageJ software. The intensity of bands for Orai1 and Orai3 were comparable in untransfected, mock transfected and scrambled shRNA-transfected BON cells.
Live cell imaging
Changes in cytosolic Ca2+ levels in live cells were performed using live-cell fluorescent imaging as described previously . Briefly, BON cells cultured on glass coverslips were loaded with a 2 μM fura-2 AM solution prepared in a physiological saline (containing in mM 140 NaCl, 5 KCl, 2.2 CaCl2, 1 MgCl2, 10 HEPES, and 5 glucose, pH 7.4) for 30–40 min at room temperature. Changes in intracellular Ca2+ were represented as the ratio of fura-2 fluorescence at 510 nm evoked by sequential excitation at 340 and 380 nm at a frequency of 1 Hz. Typically, for live cell imaging experiments changes in Ca2+ for 20–40 cells were independently monitored and analyzed. For each experiment, 2–3 coverslips were analyzed and averaged results from at least three independent experiments were used perform statistical analysis.
Mn2+ quench assay
Rate of Ca2+ entry independent of Ca2+ release or buffering was estimated by monitoring the rate of quenching of the fura-2 fluorescence signal excited at 360 nm in response to Mn2+ entry as previously described . The rate of Mn2+ quench of the fura-2 fluorescence in response to pharmacological stimulation was compared with the basal rate under the unstimulated condition. The fold change in the rates of quenching from basal values was determined.
BON cell migration was assessed as described previously using a modified Boyden chamber assay . Briefly, 5.0 × 104 BON cells were re-suspended in serum free media (SFM) and were seeded into the upper chamber, the bottom surfaces of which were coated with 50 μg/ml Type-1 rat-tail collagen (Corning Incorporated). The lower chamber was loaded with SFM containing AA with or without pharmacological inhibitors. In some experiments, the cells were pre-treated with 1 μM thapsigargin dissolved in Ca2+-free SFM for 15 min. In another experiment, the concentration of free Ca2+ was reduced to approximately 0.7 mM by adding EGTA to the media. The cells were allowed to migrate from the upper to lower chamber for 8–12 h. Following this time-period the number of migrated cells were counted and expressed as percentage of the total number seeded.
Immunofluorescence was used to identify various NMT markers in BON cells prior to or following arachidonic acid treatments. BON cells (5 × 104 cells) were seeded onto glass coverslips or Boyden chamber inserts placed in a 6-well dish containing growth media. Following treatments, cells were fixed using a 4% paraformaldehyde solution and washed with phosphate buffered saline (PBS) (Life tech). Cells were then permeabilized using a 0.5% Triton X-100 solution (Sigma) for 5 min at and a blocking buffer containing 5% non-fat dry milk added for 1 h. Following PBS washes, samples were incubated overnight at 4 °C with the appropriate primary antibody in a humidified chamber. After PBS washes, glass coverslips were incubated with secondary antibodies conjugated to Alexa Fluor 488 or Alexa Fluor 546 for 1 h and mounted with Vectashield antifade medium (Vector Laboratories). F-actin was labeled using phallotoxin conjugated to Alexa Fluor 546 or 633. Images were obtained using a Leica SP5 Confocal microscope.
The results from the live cell imaging and Mn2+-quench, were analyzed using one-way analysis of variance (ANOVA) and Dunnette’s tests were performed to assess significance among treatment groups. BON cell migration experiments using genetic and pharmacological manipulations were assessed by Tukey’s multiple comparison tests. The statistical significance of the western blot data was analyzed by 2-tailed t tests.
Store-operated and AA induced Ca2+ entry are pharmacologically distinguishable in BON cells
We next tested whether addition of AA activated a distinct Ca2+ entry pathway. Application of exogenous 1–30 µM AA induced elevations in [Ca2+] cyt in a concentration-dependent manner (Additional file 1: Figure S1). A sub-maximal dose of 6 μM AA was used to treat cells for all the experiments described in the current study. As shown in Fig. 1d, application of 6 μM AA in nominal Ca2+-containing bath solution induced an initial transient rise in [Ca2+] cyt that was followed by robust sustained elevation in [Ca2+] cyt following return of Ca2+ in the bath solution. Typically, the secondary responses were complex waveforms and we used the peak amplitude measured within the first 300 s post-application as an index of the magnitude of the AA-induced Ca2+ entry. On average in about 60% of the cells, treatment with AA resulted in Ca2+ response with bimodal kinetics, while the other 40% of cells responded with a gradual rise and sustained increase in [Ca2+] cyt . These responses were in contrast to those evoked by SOCE that showed only a transient rise in cytosolic calcium. It was unclear as to what underlies the difference between these populations. Examples of these AA-induced responses are shown in Additional file 1: Figure S1. The average change in amplitude for all the experiment was 1.48 ± 0.29 ratio units (n = 3). To further distinguish AA-induced Ca2+ entry from SOCE we assessed the effects of known pharmacological inhibitors of SOCE. As shown in Fig. 1d, e, the AA-induced rise in [Ca2+] cyt was substantially diminished by 30 μM SKF with average change in amplitude of 0.36 ± 0.06 ratio units (n = 4). In contrast, the AA-induced rise in [Ca2+] cyt was not diminished by treatment with 50 μM 2-APB and was 2.21 ± 0.12 ratio units (n = 3), demonstrating that the SOCE and the AA-induced Ca2+ entry pathways were pharmacologically distinguishable.
Arachidonic acid induced Ca2+ release from ER but did not activate SOCE
Contribution of Orai1 and Orai3 to store operated and AA-induced Ca2+ entry
In contrast to the SOCE responses, knockdown of either Orai1 or Orai3 significantly decreased the amplitude of the AA-induced responses compared to cells transfected with scrambled shRNA. While the mean amplitude of the AA-induced Ca2+ entry in cells transfected with scrambled shRNA was 1.68 ± 0.32 ratio units (n = 5), cells that were transfected with Orai1 or Orai3 shRNAs had significantly reduced peak amplitudes of 0.29 ± 0.09 (n = 6) and 0.29 ± 0.10 (n = 6) ratio units, respectively. These results indicated that both Orai1 as well as Orai3 were required for mediating Ca2+ entry in response to AA and that channels that contained predominantly Orai1 were required for SOCE.
Effects of AA-induced and store-operated Ca2+ entry on cell migration
However, because AA can be readily metabolized into other bioactive classes of compounds such as leukotrienes and prostaglandins, we assessed whether AA-metabolites contributed in BON cell migration. Moreover reports by Mohamed Trebak’s group demonstrated that leukotriene-C4 could mediate a store-independent Ca2+ entry via Orai1/3 channels [14, 24]. To test whether AA-metabolites were involved in BON cell migration, we treated the cells with pharmacological inhibitors of AA-metabolism. Ketoprofen, a non-selective inhibitor of cyclooxygenase I and II, and nordihydroguaiaretic acid (NDGA), a pan-lipoxygenase inhibitor were included along with AA in the migration assays. As shown in Fig. 6b, neither of these compounds caused a significant reduction in AA-induced cell migration. In presence of ketoprofen or NDGA, 13.7 ± 0.76% (n = 3) or 13.12 ± 0.59% (n = 3) cells migrated, respectively. These data were consistent with the idea that AA itself, and not its metabolite caused the enhancement of cell migration.
Morphological and phenotypic changes following arachidonic acid treatment
Transformed cells can exhibit a migratory phenotype that correlates with increased metastatic potential. In BON cells that express both epithelial and neuroendocrine features, this can be achieved via neuroendocrine-to-mesenchymal transition (NMT). This transition is characterized by a reduction in neuroendocrine markers and altered expression of mesenchymal proteins [32–34]. Thus, we assessed whether AA treatment could induce morphological changes and alter expression of markers of NMT including chromogranin A (CGA) E-cadherin, α-smooth muscle actin (α-SMA) and snail family transcriptional repressor 1 (Snail1).
In these experiments, cells were plated onto coverslips or applied to transwell inserts, serum starved for 4 h, incubated overnight or for up to 48 h in growth medium supplemented with 6 µM arachidonic acid or 0.1% DMSO as vehicle control and subsequently assessed by immunofluorescence. Morphological changes induced by AA treatment were visualized by F-actin staining using fluorescently labeled phalloidin. The majority of control cells appeared clustered and were rounded or oblong in shape (~ 97%), whereas the majority of AA treated cells (~ 93%) exhibited an elongated, flattened shape that was often decorated with multiple cellular processes. Additional images of these morphologies are shown in Additional file 3: Figure S3. As indicated above, migration assays revealed that AA treatment induced migration in a subset of BON cells. Therefore, we removed and imaged chamber inserts following AA treatment and assessed the morphology of non-migrated and migrated cells using confocal microscopy. As shown in Fig. 7a, b, staining for F-actin revealed distinctive morphologies between these two populations. The unmigrated cells on the surface of the transwell insert appeared to be in clusters and exhibited rounded shapes whereas the migrated cells on the bottom of the insert typically exhibited flattened irregular shapes with processes. These respective morphologies were similar to those observed for cells cultured on glass coverslips that were treated with vehicle or AA.
One hallmark of NMT is a reduction in the neuroendocrine secretory granule protein CgA. Under our experimental conditions, approximately 34% of cells in control groups exhibited perinuclear and subplasmamembrane distribution of CgA signal. Following overnight AA treatment, less than 15% of cells showed labeling. Figure 7c, d indicated that following AA treatment there was about a 50% reduction in the number of cells expressing CgA. Moreover, the treated cells appeared much more mesenchymal in morphology and some cells showed persistent expression and ubiquitous distribution of the CgA label through out the cell including the filipodial extensions. Longer treatments (24–48 h) showed a further reduction in the number of CgA expressing cells.
Furthermore, as shown in Fig. 7e, f, there was a clear reduction in the intensity of E-cadherin staining following AA treatment. In comparing images of control and AA-treated cells, there was typically greater than 60% reduction in the mean pixel intensity of the E-cadherin (green) signal. The findings were consistent with NMT phenotype. Of note, the E-cadherin signal in BON cells had an atypical nuclear distribution, similar to that previously shown to correlate with invasiveness in pancreatic NETs .
In contrast, we did not observe changes in expression levels of the mesenchymal proteins α-SMA and Snail1 following AA treatment (data not shown). The lack of an effect may be in part be explained the surprising expression of these markers in BON cells prior to AA treatment.
Previous work from our laboratory provided molecular and functional characterization of the SOCE pathway  in a variety of GEPNET cell lines. It was revealed that BON cells robustly expressed messages for Orai1 and Orai3, but not Orai2. In the current study, we extended these observations to demonstrate protein expression and functional contributions of Orai1 and Orai3 channel subunits to Ca2+ signals and cell migration. These findings support the hypothesis that AA-induced Ca2+ entry through an Orai1/Orai3 containing channel was important for migration of a sub-population of BON cells.
Recent work from several groups has demonstrated a requirement of Orai1-containing channels for SOCE and cell migration in some tumor cell lines. Although knockdown of Orai1 suppressed BON cell migration, it was unlikely that the suppression was mediated by inhibition of a canonical store-operated channel. This idea was supported by the observation that direct activation of SOCE by TSG-induced store depletion was ineffective at stimulating migration. Although this finding is tempered by the caveat that prolonged treatment with TSG can induce ER stress and apoptosis.
Our contention is that it was more likely that the knockdown of Orai1 inhibited an AA-induced Ca2+ entry channel that contained Orai3 as well as Orai1 subunits. Consistent with this proposal, we found that treatment with AA resulted in Ca2+ influx that was distinct from canonical SOCE. Moreover, activation of this pathway stimulated migration of BON cells, and that when inhibited, abrogated the enhancement in migration. These data showed that Orai1 was necessary for mediating SOCE, whereas AA-induced Ca2+ entry as well as BON cell migration required both Orai1 and Orai3.
The AA-induced Ca2+ entry channel characterized here is reminiscent of the store-independent ARC channel described by Shuttleworth’s group [35, 36]. More recently, Trebak’s group identified an Orai1/Orai3-containing leukotriene-C4 (LTC4) inducible channel that mediated Ca2+ entry in vascular smooth muscle cells in response to thrombin stimulation . Although we did not directly test whether LTC4 could activate Ca2+ entry or cell migration in our system, pharmacological inhibition of AA-metabolism did not suppress AA-induced cell migration. This indicated that the enhancement of cell migration observed was largely due to AA itself and not its metabolite.
Assessment of migration using pharmacological tools was also consistent with the involvement of an Orai3-containing channel. Typically, 2-APB has been used as a tool to block SOCE and has been shown in some studies to inhibit cell migration. For example, 2-APB was shown to block EGF-induced cell migration in nasopharyngeal carcinoma  and wound healing of clear cell renal cell carcinoma . However, we found that treatment with 2-APB suppressed SOCE but not AA-induced Ca2+ entry or AA-evoked BON cell migration, consistent with previous work that showed 2-APB does not block, and may even enhance, conductance in Orai3-containing channels. In contrast, the broader spectrum channel blocker SKF effectively blocked Ca2+ entry through Orai1- and Orai3-containing channels. These findings indicated a novel mechanism by which Orai-mediated Ca2+ entry may contribute to migration in this biologically idiosyncratic group of cancers.
In this context, it should be noted that a mutual antagonism between SOCE- and AA-evoked Ca2+ entry has been observed in other cell types [37–39] and thus modes of entry may interact to set the migration potential of NET cells. Indeed, the migration potential may in part be mediated by NMT, consistent with our findings that AA treatment altered the morphology and phenotype of BON cells such that the migrated cells appeared more mesenchymal in state.
Based on our findings it is tempting to speculate that the AA-induced Ca2+ channel has relevance for GEPNET pathophysiology. For example, these cancers are thought to arise from neurosecretory cells in the gastrointestinal tract and commonly metastasize to the liver. Both the small intestine and liver exhibit elevated concentrations of AA [40, 41] compared to other regions of the gut. Moreover, neuroendocrine tumor cells are known to secrete several biogenic amines and peptides. Many of these secretory products have been linked with increased production of AA in different cell types. For example, serotonin has been shown to stimulate the PLA2-driven synthesis of AA in hippocampal neurons . Bradykinin, an intestinal peptide that is secreted by neuroendocrine cells has been shown to stimulate AA synthesis in gut . Moreover, we recently reported that conditioned media (CM) derived from cultured BON cells had a pro-migratory effect on these tumor cells, presumably through an autocrine mechanism . Although we did not directly, test whether treatment with CM stimulated synthesis of AA in our system, unpublished experiments related to this study showed that the pharmacological inhibition of AA synthesis resulted in a decrease in CM-evoked cell migration that could be recovered by application of AA.
The current manuscript demonstrates that treatment with AA induced Ca2+ entry through an Orai1/Orai3 heteromeric ion channel and that this mode of Ca2+ entry is important for migration in a GEPNET cell-line. The findings suggest that AA-induced Ca2+ entry may help set the migratory potential of these tumor cells and identify Ca2+ entry through the Orai3-containing channel as a novel signal for BON cell migration that may be exploited for therapeutic prevention of recurring GEPNET metastasis.
PG designed experiments, acquired and analyzed data and wrote the manuscript. TP performed experiments and analyzed immunofluorescence data. DG conceived and directed the research, reviewed the data and wrote and edited the manuscript. All authors reviewed the manuscript. All authors read and approved the final manuscript.
We thank Dr. Andrea Kalinoski and the Advanced Microscopy and Imaging Center at University of Toledo for assistance with the confocal microscopy.
The authors declare that they have no competing interests.
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The data used and/or analysed for the current study are available from the corresponding author on reasonable request.
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This study was supported by a grant from the Raymond and Beverly Sackler Foundation to DRG and University of Toledo Graduate Student Research award to PG.
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