NCS‐1 expression is higher in basal breast cancers and regulates calcium influx and cytotoxic responses to doxorubicin

Neuronal calcium sensor‐1 (NCS‐1) is a positive modulator of IP3 receptors and was recently associated with poorer survival in breast cancers. However, the association between NCS‐1 and breast cancer molecular subtypes and the effects of NCS‐1 silencing on calcium (Ca2+) signaling in breast cancer cells remain unexplored. Herein, we report for the first time an increased expression of NCS‐1 in breast cancers of the basal molecular subtype, a subtype associated with poor prognosis. Using MDA‐MB‐231 basal breast cancer cells expressing the GCaMP6m Ca2+ indicator, we showed that NCS‐1 silencing did not result in major changes in cytosolic free Ca2+ increases as a result of endoplasmic reticulum Ca2+ store mobilization. However, NCS‐1 silencing suppressed unstimulated basal Ca2+ influx. NCS‐1 silencing in MDA‐MB‐231 cells also promoted necrotic cell death induced by the chemotherapeutic drug doxorubicin (1 µm). The effect of NCS‐1 silencing on cell death was phenocopied by silencing of ORAI1, a Ca2+ store‐operated Ca2+ channel that maintains Ca2+ levels in the endoplasmic reticulum Ca2+ store and whose expression was significantly positively correlated with NCS‐1 in clinical breast cancer samples. This newly identified association between NCS‐1 and basal breast cancers, together with the identification of the role of NCS‐1 in the regulation of the effects of doxorubicin in MDA‐MB‐231 breast cancer cells, suggests that NCS‐1 and/or pathways regulated by NCS‐1 may be important in the treatment of basal breast cancers in women.


Introduction
Aberrations in calcium (Ca 2+ ) signaling and associated regulatory proteins such as Ca 2+ channels occur in a variety of cancers (Stewart et al., 2015). Cancer cells may remodel their intracellular Ca 2+ signaling machinery to favor tumorigenic processes that enable continued survival and proliferation . For example, prostate cancers exhibit increased expression of Ca 2+ channels such as transient receptor potential vanilloid 6 (TRPV6) (Fixemer et al., 2003) and ORAI3 channels (Dubois et al., 2014) compared to normal prostate tissues. The remodeling of ORAI3 channel expression in cancer cells causes a shift in Ca 2+ influx from a store-regulated mechanism normally used by healthy prostate cells toward a store-independent one that confers apoptotic resistance and proliferative signaling (Dubois et al., 2014). Specific remodeling of Ca 2+ signaling is evident during tumor progression and is also seen between different cancer subtypes such as in breast cancer. Expression of the ORAI1 Ca 2+ influx channel is higher in breast cancers of the basal molecular subtype, which are often triplenegative (i.e., do not express estrogen, progesterone, and HER2 receptors) compared to nonbasal breast cancers (McAndrew et al., 2011). Conversely, luminal breast cancer cell lines generally have higher ORAI3 expression and exhibit greater ORAI3-mediated Ca 2+ entry compared to basal breast cancer cell lines (Motiani et al., 2010;Motiani et al., 2013).
Studies of Ca 2+ signaling in cancer cells have usually focused on plasma membrane Ca 2+ permeable ion channels and their regulators (Deliot and Constantin, 2015;Vashisht et al., 2015). In contrast, far fewer studies have assessed the potential remodeling of proteins regulating endoplasmic reticulum (ER) Ca 2+ homeostasis in cancer. The ER is the main intracellular store for releasable Ca 2+ in response to activating stimuli. The major proteins regulating ER Ca 2+ homeostasis in epithelial cells are the sarco/ER Ca 2+ ATPase (SERCA) pumps, which actively sequester cytosolic Ca 2+ into the ER lumen, and inositol triphosphate receptors (IP3R), which release ER Ca 2+ in response to IP 3 -mobilizing agonists such as ATP. The release of ER Ca 2+ through IP3R activates an ER-refilling mechanism called store-operated Ca 2+ entry (SOCE). During SOCE, an ER-resident Ca 2+ sensor protein called stromal interaction molecule 1 (STIM1) senses the reduced ER Ca 2+ levels and oligomerizes to form a Ca 2+ -permeant protein complex with ORAI channels on the plasma membrane, facilitating Ca 2+ influx. Dysregulation in ER Ca 2+ homeostasis is associated with some cancerous phenotypes including the ability to resist apoptosis and the promotion of prosurvival signaling, which can influence response to anticancer therapies (Pedriali et al., 2017). Abnormal SERCA expression and/or activity is associated with some cancers including colorectal (Fan et al., 2014;Yang et al., 2015) and blood cancers (Roti et al., 2013). These alterations could be targeted for cancer treatment. Indeed, mipsagargin, a prostate-specific membrane antigen-based prodrug targeting the SERCA pumps, is currently in clinical trials for the treatment of solid tumors (Mahalingam et al., 2016). Alterations in ER Ca 2+ signaling can also be mediated through protooncogenes such as the Bcl-2 family of antiapoptotic proteins, which lower ER Ca 2+ levels by increasing ER Ca 2+ 'leak' (Bittremieux et al., 2016;Foyouzi-Youssefi et al., 2000). Apart from these more well-defined mechanisms of ER Ca 2+ store regulation, there are also proteins which indirectly modulate ER Ca 2+ signals such as the neuronal calcium sensor-1 (NCS-1).
Neuronal calcium sensor-1 is a 22-kDa high-affinity EF-hand-containing Ca 2+ sensor protein with structural similarity to calmodulin. NCS-1 contains an Nterminal myristoyl group that allows Ca 2+ -dependent binding to proteins and cellular membranes. NCS-1 interacts with IP3R and increases its opening probability (Schlecker et al., 2006), and NCS-1 silencing reduces IP 3 -mediated Ca 2+ signals mediated by ATP in neuroblastoma cells (Boehmerle et al., 2007) and endothelin in cardiomyocytes (Zhang et al., 2010). NCS-1 is widely expressed in adult neuronal cells (Nakamura and Wakabayashi, 2012;Weiss et al., 2010) and is involved in the regulation of neurotransmission activity important for learning, memory, and synaptic plasticity (Sippy et al., 2003;Weiss et al., 2010). NCS-1 also regulates Akt-dependent prosurvival signaling in neurons (Nakamura et al., 2006) and cardiomyocytes (Nakamura et al., 2016). NCS-1 is implicated in a variety of disease states such as schizophrenia and bipolar disorder (Boeckel and Ehrlich, 2018;Koh et al., 2003). In the context of cancer, NCS-1 is a potential target for the prevention of paclitaxel-induced peripheral neuropathy (Mo et al., 2012). Ehrlich et al. showed that paclitaxel treatment enhances the binding of NCS-1 to IP3R in neuronal cells  and prolonged paclitaxel treatment results in dysregulation of IP 3 -dependent Ca 2+ signaling due to the degradation of NCS-1 via Ca 2+ -mediated calpain activation (Boehmerle et al., 2007). This dysregulated Ca 2+ signaling was proposed to cause the peripheral neuropathy induced by paclitaxel. More recently, NCS-1 was shown to play a role in breast cancer invasion and migration in vitro, and higher NCS-1 expression correlates with poorer survival in breast cancer patients (Moore et al., 2017).
Despite the involvement of NCS-1 in regulating Ca 2+ homeostasis and its association with breast cancer, no studies have assessed the role of NCS-1 in intracellular Ca 2+ signaling in breast cancer cells. The association of NCS-1 expression with breast cancer molecular subtypes also remains unexplored. Here, we report for the first time that NCS-1 expression is increased in the basal breast cancer molecular subtype. We also demonstrate that siRNA-mediated silencing of NCS-1 attenuated unstimulated basal Ca 2+ influx in basal breast cancer cells. Silencing NCS-1 enhanced doxorubicin-induced breast cancer cell death, which was a phenomenon phenocopied by the silencing of the Ca 2+ store refilling channel ORAI1. These studies highlight a potentially important role for NCS-1 in the regulation of Ca 2+ influx pathways important in the induction of cell death by some therapies in basal breast cancer cells.
A gene expression heatmap was generated using gene expression data from the TCGA database. The TCGA gene expression data were mean-centered and hierarchically clustered using Multiple Experiment Viewer (v4.8.1; Saeed et al., 2003) with Manhattanbased average-linkage clustering. Displayed above the heatmap are the PAM50 molecular subtypes. Molecular markers typical of the different molecular subtypes were used in the clustering.

Patient survival analysis in basal breast cancer
Overall patient survival in basal breast cancers based on NCS-1 expression was assessed using the Kaplan-Meier Plotter web-based tool (Gyorffy et al., 2010). Affymetrix probe IDs used in the analyses were the mean of 230146_s_at, 222570_at, and 238753_at, and high or low NCS-1 expression was stratified using the 'auto-select best cutoff' function. This function assesses median, tertile, and quartile expression cutoffs and utilizes the best cutoff for the given gene and dataset. Hazard ratio and log-rank P values are shown in the figure.

Gene correlation analysis
Gene correlation analyses were performed on the R2 Genomics Visualization Platform (http://r2.amc.nl) using TCGA microarray datasets. Correlation coefficients between NCS-1 and assessed genes are shown as R-values and significance of correlations is shown as P-values.

NCS-1 lentiviral production and transduction
Human NCS-1 was amplified from Applied Biological Material (LV800666) plasmid by PCR and cloned into the pCDH-EF1-FHC lentiviral vector (Addgene # 64874). Lentiviral particles were produced in HEK293T cells with second-generation packaging plasmids and Lipofectamine 3000 transfection. The medium containing viral particles was collected after 48 h. GCaMP6m-MDA-MB-231 cells were subsequently transduced with the empty vector (EV) or human NCS-1 in the presence of polybrene (8 µgÁmL À1 ). The viral media were replaced with fresh media 24 h after infection and cells were selected with puromycin (2 µgÁmL À1 ) 48 h after viral infection.

Calcium imaging using fluorometric imaging plate reader (FLIPR)
Cells were seeded into black-walled 96-well plates (Corning Incorporated, Corning, NY, USA) at a density of 4 9 10 3 cells per well and Ca 2+ imaging was performed 96 h post-siRNA transfection using the FLIPR TETRA (Molecular Devices, San Jose, CA, USA). GCaMP6m-MDA-MB-231 cells with exogenous overexpression of NCS-1 or expressing the EV control were seeded at a density of 1 9 10 4 cells per well, and Ca 2+ imaging was performed 72 h after seeding. Briefly, media was removed from the wells, washed once, and then replaced with physiological salt solution (PSS, composed of 10 mM HEPES, 5.9 mM KCl, 1.4 mM MgCl 2 , 1.2 mM NaH 2 PO 4 , 5 mM NaHCO 3 , 140 mM NaCl, 11.5 mM glucose, pH 7.2) containing nominal Ca 2+ (no added CaCl 2 ) and incubated for 15 min at room temperature prior to Ca 2+ -imaging studies. For the assessment of ER Ca 2+ signaling, IP 3 -mobilizing agents (ATP; Sigma-Aldrich) at 1, 3, and 100 µM and trypsin (Sigma-Aldrich) at 1, 10, and 100 nM concentrations) and cyclopiazonic acid (CPA; Sigma at 10 µM concentration) prepared in PSS were added to wells. To assess unstimulated Ca 2+ influx, 1.8 mM CaCl 2 was added. A 100 µM concentration of bis(2-aminophenoxy)ethane tetraacetic acid (BAPTA; Invitrogen TM ) was included during the addition of reagents. For assessment of SOCE, BAPTA (100 µM) was first added to cells preincubated for 15 min in PSS nominal, followed by CPA (10 µM) addition to mediate ER store depletion. CaCl 2 (1.8 mM) was then added after 700 s to facilitate SOCE. An ORAI1 inhibitor Synta66 (10 µM; Sigma) was included in experiments assessing basal Ca 2+ influx and SOCE. Changes in fluorescence intensity relative to baseline fluorescence over time were expressed as cytosolic Ca 2+ changes (DF/F o ) and were measured at 470-495 nm excitation and 515-575 nm emission wavelengths and analyzed using the SCREENWORKS Software (Molecular Devices).

Assessment of cell proliferation and death using epifluorescence microscopy
Cell proliferation was assessed using the Click-iT TM EdU Alexa Fluor TM 555 imaging kit (Invitrogen). Briefly, 24 and 48 h after siRNA transfection and doxorubicin (24 h) treatments, cells were incubated with EdU (10 µM) for 1 h at 37°C. Cells were then fixed with 4% paraformaldehyde for 15 min, washed with PBS containing 3% BSA, and permeabilized using 0.5% Triton-X for 20 min. Cells were then incubated in the dark with the Click-iT reaction cocktail (containing Alexa Fluor azide) for 1 h, prepared according to manufacturer's instructions. Cell nuclei were stained using Hoecsht 33342 (Invitrogen, Vista, CA, USA; 400 nM).
For the assessment of necrotic cell death, at 24 h post-siRNA transfection, medium was replaced with 10% FBS and HEPES-buffered Fluorobrite TM DMEM (Invitrogen) or Fluorobrite media containing 0.03 or 1 µM doxorubicin (Merck Millipore, MilliporeSigma, Burlington, MA, USA) and incubated for 24 h before replacing with drug-free Fluorobrite DMEM. After 72 h, cells were stained with Hoechst 33342 (10 µgÁmL À1 ; Invitrogen) and propidium iodide (PI; 1 µgÁmL À1 ; Invitrogen). Cells for both proliferation and death experiments were imaged using the ImageXpress Micro (Molecular Devices) epifluorescence microscope, using the DAPI and Cy3 filter sets to assess EdU-positive cells or PI-positive cells as described previously (Curry et al., 2012;Peters et al., 2012). The multiwavelength cell scoring module (MetaXpress 6.0) was used to assess the percentage of EdU-positive or PI-positive cells.

Statistical analysis
Statistical analyses of individual experiments were performed using GRAPHPAD PRISM (version 7; GraphPad Software, San Diego, CA, USA) as described in the corresponding figure legends. Data are presented as mean AE SEM (of three independent experiments).

NCS-1 expression is higher and is predictive of poorer survival in the basal breast cancer molecular subtype
Given that NCS-1 was recently reported to be associated with increased breast tumor aggression and poor prognosis (Moore et al., 2017), we explored if NCS-1 is associated with any of the breast cancer intrinsic molecular subtypes using TCGA breast cancer database (Cancer Genome Atlas, 2012). Hierarchical clustering of NCS-1 and breast cancer molecular markers showed a positive correlation with basal and proliferative markers, such as KRT5, 14 and 17, CDH3, FOXC1, FOXM1, EGFR, and MKI67 (Fig. 1A). Conversely, NCS-1 expression is negatively correlated to FOXA1, ESR1, and PGR, which are more commonly associated with the breast cancer luminal subtype. We found that NCS-1 expression is also significantly higher in the basal molecular subtype (Fig. 1B) compared to other breast cancer subtypes. NCS-1 levels were also highest in the basal-like immune-activated (BLIA) and basal-like immune-suppressed (BLIS) subtypes within the triple-negative breast cancer (TNBC) subtypes (Burstein et al., 2015) (Fig. 1C). We also observed a trend toward increased NCS-1 expression in basal breast cancer cell lines compared to HER2 and luminal breast cancer cell lines (Fig. 1D).
Finally, to assess if NCS-1 expression had any association with patient survival in basal breast cancers, we used the Kaplan-Meier Plotter online tool to stratify the overall survival (OS) of breast cancer patients with basal tumors based on NCS-1 gene expression (Gyorffy et al., 2010). As shown in Fig. 1E, higher NCS-1 expression correlates with a poorer OS in the basal breast cancer patient subgroup (HR = 3.15, P = 0.0002), further implicating the significance of NCS-1 in basal breast cancers.
3.2. NCS-1 silencing has no major effect on ER Ca 2+ signaling but suppresses unstimulated, basal Ca 2+ influx in MDA-MB-231 cells Due to the role of NCS-1 as a positive regulator of IP3Rs (Boehmerle et al., 2007;Schlecker et al., 2006) and the lack of studies defining a role for NCS-1 in Ca 2+ signaling in breast cancer cells, we assessed two possible consequences of NCS-1 silencing in the GCaMP6m-MDA-MB-231 breast cancer cell line. These two potential consequences reduced IP 3 -mediated Ca 2+ store release after G-protein-coupled receptor activation (Berridge, 2016) and reduced compensatory basal Ca 2+ influx as a result of less basal ER Ca 2+ leak from IP3Rs (Mignen et al., 2017). We inhibited NCS-1 expression using siRNAs ( Fig. 2A-C) and assessed relative intracellular [Ca 2+ ] CYT increases in response to ATP and trypsin. In the absence of extracellular Ca 2+ , ATP and trypsin addition mobilizes ER Ca 2+ stores through an IP 3 -mediated pathway. NCS-1 silencing had no significant effect on [Ca 2+ ] CYT increases mediated by any concentration of ATP (Fig. 2D,E) and trypsin at 1 and 10 nM (Fig. 2F,G) concentrations; however, NCS-1 silencing modestly suppressed Ca 2+ signals in response to trypsin at 100 nM (Fig. 2G). [Ca 2+ ] CYT increases induced by SERCA inhibition using CPA were also not significantly affected by NCS-1 silencing (Fig. 2H,I). Collectively, these data suggest that NCS-1 is not a major regulator of ER Ca 2+ release in MDA-MB-231 breast cancer cells.
In some cancer cells, altered Ca 2+ influx in the absence of external stimuli (unstimulated or basal Ca 2+ influx) is associated with key tumorigenic traits, such as increased proliferation and migration (Chantome et al., 2013;Feng et al., 2010;Mignen et al., 2017;Peters et al., 2012). Thus, we next investigated the effect of NCS-1 silencing on unstimulated, basal Ca 2+ influx. As shown in Fig. 3A, when extracellular Ca 2+ (1.8 mM) was added to GCaMP6m-MDA-MB-231 cells incubated in nominal Ca 2+ conditions, the increase in [Ca 2+ ] CYT levels induced by the readdition of extracellular Ca 2+ was attenuated when NCS-1 was silenced. Analysis of the peak (Fig. 3B) and rate of Ca 2+ influx from 9 to 100 s (Fig. 3C) revealed a significant decrease in Ca 2+ influx with NCS-1 silencing. To assess if NCS-1 silencing also reduced Ca 2+ influx through SOCE, we performed a classical SOCE experiment using CPA-mediated ER Ca 2+ store depletion, followed by Ca 2+ readdition. We first confirmed that silencing of the store-operated Ca 2+ channel ORAI1 ( Fig. 3D-F) eliminated SOCE (Fig. 3G,H). However, silencing of NCS-1 had no effect on SOCE (Fig. 3G,  H). We have previously shown that unstimulated Ca 2+ influx occurs through ORAI1 in HC11 mammary epithelial cells . To assess if unstimulated Ca 2+ influx occurred through ORAI1 in GCaMP6m-MDA-MB-231 cells, we assessed the effect of ORAI1 silencing. As shown in Fig. 3I,J, ORAI1 silencing suppressed unstimulated Ca 2+ influx. Collectively, these results identify a critical role for NCS-1 in modulating unstimulated Ca 2+ influx likely through ORAI1 channels, since ORAI1 silencing phenocopied the effect of NCS-1 silencing in GCaMP6m-MDA-MB-231 breast cancer cells.

NCS-1 overexpression reduces ATP-induced Ca 2+ release but does not affect unstimulated Ca 2+ influx
In light of the observed role of NCS-1 silencing on unstimulated Ca 2+ influx, we further investigated if this Ca 2+ influx pathway could be enhanced with NCS-1 overexpression. We generated stable NCS-1-overexpressing GCaMP6m-MDA-MB-231 cells (NCS1-OE) using lentiviral transduction with a commercially available human NCS-1 plasmid (Fig. 4A). We first assessed the functional role of NCS1-OE cells in IP 3mediated ER Ca 2+ release using ATP, and showed that NCS1-OE cells reduced ER Ca 2+ release in response to 100 µM ATP (Fig. 4B,C) compared to GCaMP6m-MDA-MB-231 cells expressing the EV control. We then assessed unstimulated Ca 2+ influx in NCS1-OE cells compared to EV cells. As shown in Fig. 4D,E, NCS-1 overexpression did not enhance unstimulated Ca 2+ influx in GCaMP6m-MDA-MB-231 cells. Unstimulated Ca 2+ influx was inhibited with the addition of the ORAI1 inhibitor, Synta66 (Fig. 4D,E). NCS-1 overexpression also did not have any significant effect on SOCE (Fig. 4F,G). Collectively, these results demonstrate that NCS-1 is not a major direct regulator of SOCE and that promotion of unstimulated Ca 2+ influx may already be maximal in GCaMP6m-MDA-MB-231 breast cancer cells.

Correlation between NCS-1 expression and Ca 2+ channels or pumps
Given the ability of NCS-1 silencing to phenocopy the effects of ORAI1 silencing in MDA-MB-231 breast cancer cells, we explored the possibility that NCS-1 silencing-mediated suppression of unstimulated Ca 2+ influx was due to reduced expression of ORAI1. As shown in Fig. 5A, ORAI1 mRNA levels were affected neither by NCS-1 silencing nor by the expression of its regulators, STIM1 and STIM2. The expression of the major ER Ca 2+ regulators, IP 3 R and SERCA pumps, were also unaffected by NCS-1 silencing (Fig. 5B). We also assessed the correlation between NCS-1 and Ca 2+ influx channels using TCGA breast cancer patient datasets on the R2 Genomics Visualization Platform as shown in Fig. 5C. Among the assessed genes, the most positively correlated genes with NCS-1 were TRPV6 (R = 0.392), TRPM8 (R = 0.340), and ORAI1 (R = 0.23), whereas the most negatively correlated genes were ORAI3 (R = À0.376) and TRPM7 (R = À0.296).

NCS-1 and ORAI1 silencing enhances percentage of cell death with doxorubicin treatment
Despite reports of NCS-1 being associated with therapeutic response to paclitaxel (Moore et al., 2017; Fig. 1. NCS-1 expression is correlated with the basal breast cancer molecular subtype. (A) Clustered heatmap of normalized RNA-Seq expression data, where low expression is indicated in blue and high expression in red. Data are log2 mean-centered RSEM values sourced from TCGA. Displayed on the right are the Pearson's correlation coefficients of NCS1 expression versus the molecular markers, and corresponding P-values are indicated. Indicated above the heatmap are the PAM50 molecular subtypes for each breast tumor. (B) Relative gene expression of NCS-1 in breast tumors from TCGA database (Cancer Genome Atlas, 2012) categorized according to breast cancer molecular subtypes. Statistical analysis was performed using a one-way ANOVA with Tukey's test. ****P < 0.0001, ***P < 0.0002. (C) Relative gene expression levels of NCS-1 in TNBC subtypes as described by Burstein et al. (2015). NCS-1 expression is higher in BLIA and BLIS compared to MES and LAR subtypes. Data were downloaded from R2 Genomics Analysis Platform and plotted in GRAPHPAD PRISM. Statistical analysis was performed using a one-way ANOVA with Tukey's test. ***P < 0.001, * P < 0.05. (D) NCS-1 expression in 40 breast cancer cell lines grouped to the molecular subtypes and in 4 nontumorigenic, breast epithelial cell lines. Data obtained from Array Express (accession number E-MTAB-181) (Heiser et al., 2012). (E) OS of patients with basal breast cancer stratified to NCS-1 expression. Data were sourced from the Kaplan-Meier Plotter online tool (Gyorffy et al., 2010) Moore et al., 2018), it remains unknown if this is associated with changes in Ca 2+ signaling in breast cancer cells. Chemosensitization through the suppression of Ca 2+ influx via inhibition of SOCE has been reported in pancreatic and liver cancer models (Kondratska et al., 2014;Tang et al., 2017). We therefore assessed if ORAI1 and NCS-1 silencing both augment the effects of doxorubicin, a commonly used chemotherapy in the treatment of basal or TNBCs. We first assessed if silencing of NCS-1 and ORAI1 promoted the antiproliferative effects of doxorubicin using EdU staining. As shown in Fig. 6A, NCS-1 and ORAI1 silencing alone did not alter proliferation of MDA-MB-231 cells under these conditions. NCS-1 and ORAI1 silencing also did not augment antiproliferative effects of doxorubicin at 24 h (Fig. 6B,D) or 48 h (Fig. 6C,E). We next assessed the potential of NCS-1 silencing to promote cell death induced by doxorubicin using PI staining. As shown in Fig. 7A,B, both NCS-1 and ORAI1 silencing alone did not induce cell death. However, with doxorubicin treatment, NCS-1 silencing significantly enhanced the percentage of PI-positive cells induced by doxorubicin (1 µM) treatment (Fig. 7A). This increase in doxorubicin-(1 µM)-induced cell death was phenocopied by ORAI1 silencing (Fig. 7B).
We further investigated if the promotion of cell death with NCS-1 and ORAI1 silencing is a result of siRNA-mediated inhibition of ORAI1 but not NCS-1 suppresses SOCE, that is, Peak 2 during Ca 2+ readdition at 750 s. Statistical analysis was performed using a one-way ANOVA with Bonferroni's posthoc test. ****P < 0.001 (I) Trace shows the Ca 2+ readdition phase of wells not pretreated with CPA from the same experiment shown in (G) from 300 to 1800 s. The trace shows a reduction in unstimulated Ca 2+ influx as a result of ORAI1 silencing. (J) Bar graph shows the mean AE SEM of the maximal [Ca 2+ ] CYT increases during Ca 2+ readdition from 700 to 1800 s. Statistical analysis was performed using a paired t-test. *P < 0.05; **P < 0.002, ****P < 0.0001.  increased apoptotic cell death by assessing PARP-1 cleavage. As shown in Fig. 7C-E, although doxorubicin treatment resulted in a concentration-dependent increase in PARP cleavage, both NCS-1 and ORAI1 silencing did not promote PARP cleavage at any concentration. The lack of the effect of NCS-1 silencing on promoting apoptotic cell death was also observed with paclitaxel (Fig. 7F,G).

Discussion
Neuronal calcium sensor-1 is a recently identified negative prognostic indicator for breast cancer (Moore et al., 2017). In this study, we report for the first time that NCS-1 is more highly expressed in the basal molecular subtype, a subtype which has a strong overlap with TNBC and is associated with poorer survival rates in breast cancer patients (Prat et al., 2015;Rivenbark et al., 2013;Sorlie et al., 2003). We also observed that higher levels of NCS-1 predict poorer survival within the basal molecular breast cancer subtype. The association between increased NCS-1 expression and basal-like subtypes was also supported in our assessment of the more recently identified TNBC molecular subtypes (Burstein et al., 2015), since higher levels of NCS-1 was seen in both the BLIA and the BLIS subtypes compared to the mesenchymal (MES) and the luminal androgen receptor (LAR) subtypes. Moreover, a positive correlation between NCS-1 gene expression and genes typically associated with a basal molecular signature was observed (Prat et al., 2015). Our identification that NCS-1 is most associated with the basal breast cancer further defines the potential subtype-specific contribution of NCS-1 to breast cancer progression.
Despite the recently reported association between NCS-1 and increased breast cancer invasion and migration (Apasu et al., 2019;Moore et al., 2017), the role of NCS-1 in Ca 2+ signaling in breast cancer cells has not been reported. IP 3 -mediated Ca 2+ signaling is initiated by a variety of proproliferative and promigratory receptors (Mound et al., 2017;Szatkowski et al., 2010) and could be predicted to be augmented as a consequence of elevated levels of the IP3R-positive modulator NCS-1 in basal breast cancers. Given increased activity of IP3Rs with NCS-1 and the recent report that cardiac cells isolated from NCS-1 knockout mice exhibit reduced Ca 2+ transients with ATP stimulation (Nakamura et al., 2011), we hypothesized that NCS-1 silencing in MDA-MB-231 cells would suppress Ca 2+ mobilization induced by ATP via purinergic receptors and also by trypsin, which activates  protease-activated receptors (Mari et al., 1996). However, there was no major change in IP 3 -mediated Ca 2+ signals with NCS-1 silencing in MDA-MB-231 breast cancer cells. Only a modest but statistically significant reduction in the [Ca 2+ ] CYT increase induced by 100 nM trypsin was observed. The lack of a major effect on ER Ca 2+ release after G-protein-coupled receptor activation could be explained by a compensatory response, such as an upregulation of components of IP 3 -mediated Ca 2+ store release. Analogous to such a change is the upregulation of IP3R1 in mouse embryonic fibroblasts deficient in presenilins (Kasri et al., 2006), proteins that have also been proposed to promote the loss of Ca 2+ from the ER (Tu et al., 2006). However, our studies found no change in IP3R or SERCA mRNA levels as a consequence of NCS-1 silencing. These observations suggest that the role and function of NCS-1 in ER Ca 2+ release in MDA-MB-231 cells are not straightforward. Indeed, studies in neuronal cells reported that NCS-1 binds to different Ca 2+ regulators, which are important for the fine-tuning of Ca 2+ signals regulating specific processes such as neurite elongation and branching (Hui et al., 2007;Hui et al., 2006;Iketani et al., 2009).There have been no studies assessing the effect of NCS-1 silencing on Ca 2+ signaling in MDA-MB-231 cells despite two consecutive studies demonstrating a role for NCS-1 in promoting tumor cell migration and aggressiveness in the same cell line (Apasu et al., 2019;Moore et al., 2017). Based on our studies, it therefore appears that NCS-1 silencing can modulate cell death and perhaps migration (Apasu et al., 2019;Moore et al., 2017) in MDA-MB-231 cells with only a modest or even no effects on activated IP 3 -mediated Ca 2+ release.
An alternative explanation is that basal breast cancer cells with elevated NCS-1 have an upregulation of unstimulated basal Ca 2+ influx, perhaps to maintain ER Ca 2+ store levels or via another mechanism. ER Ca 2+ leak via IP 3 Rs occurs in a variety of cell types (Bittremieux et al., 2016;Boutin et al., 2015). Consistent with this hypothesis, we found that NCS-1 silencing suppressed basal Ca 2+ influx in MDA-MB-231 cells. One obvious mechanism for such a compensatory pathway is the store-operated Ca 2+ channel ORAI1 which is activated by lowered ER Ca 2+ levels (Brandman et al., 2007;Subedi et al., 2018). ORAI1 is implicated in basal Ca 2+ influx important for maintaining ER Ca 2+ homeostasis (Brandman et al., 2007;Zuccolo et al., 2018) in a variety of cell types, although other channels including TRPV6 are also implicated in basal Ca 2+ influx (Lehen'kyi et al., 2007;Peng et al., 1999;Peters et al., 2012). However, MDA-MB-231 cells have negligible (Peters et al., 2012) or undetectable levels of TRPV6 (G.R. Monteith & D. McAndrew, unpublished data), so this compensation is likely to be via the ORAI1 channel in this model. Indeed, the silencing of ORAI1 phenocopied the suppression of unstimulated Ca 2+ influx by NCS-1 silencing in our studies. The lack of change in ORAI1, STIM1, and STIM2 mRNA levels with NCS-1 silencing suggests that nontranscriptional mechanisms such as altered trafficking or activity are involved. It could simply be the case that the reduced loss of Ca 2+ from the ER with NCS-1 silencing is detected immediately by STIM1 or STIM2 resulting in less ORAI1 activation in the resting cells and, in turn, reducing the compensatory basal Ca 2+ influx. Such a mechanism is supported by the lack of effect of NCS-1 silencing on maximally activated SOCE. Alternatively, ORAI1 can be regulated by phosphoinositides (Calloway et al., 2011;Walsh et al., 2009). NCS-1 silencing could thus suppress ORAI1-mediated basal Ca 2+ influx via this mechanism, as NCS-1 was shown to regulate phosphoinositide remodeling in PC12 rat adrenal cells (Koizumi et al., 2002). Our studies also do not completely discount the possibility that NCS-1 regulates other Ca 2+ influx channels, such as voltage-gated Ca 2+ channels or TRP channels as observed in presynaptic neuronal cells (Yan et al., 2014) and PC12 cells (Hui et al., 2006). These mechanisms should be explored in future studies assessing the link between NCS-1 and Ca 2+ signaling in cancer cells.
Although exogenous NCS-1 overexpression could be predicted to promote unstimulated Ca 2+ influx, since silencing of NCS-1 inhibited this Ca 2+ influx pathway, our studies using NCS-1 overexpressing cells did not show this result. This suggests that NCS-1 levels in MDA-MB-231 may already maximally activate unstimulated Ca 2+ influx. In this context, Moore et al. reported that silencing of NCS-1 in MDA-MB-231 suppressed wound closure but NCS-1 overexpression in MDA-MB-231 cells did not promote wound closure (Moore et al., 2017). Hence, NCS-1 may already have a maximal contribution to a variety of processes in MDA-MB-231 cells at endogenous expression levels. Given the established promigratory role of ORAI1 in MDA-MB-231 breast cancer cells (Yang et al., 2009), and our identified potential link between ORAI1-mediated unstimulated Ca 2+ influx and NCS-1, the role of ORAI1 on the effects of NCS-1 silencing on MDA-MB-231 breast cancer cell migration should be assessed in future studies.
Further association between NCS-1 and ORAI1 was seen in breast cancer samples where a positive correlation was observed between these two genes. NCS-1 was also significantly positively correlated with the aforementioned channel TRPV6 that is associated with unstimulated Ca 2+ influx in many cells of epithelial origin (Lehen'kyi et al., 2007;Peters et al., 2012). These data suggest that NCS-1-overexpressing breast cancer cells may compensate for enhanced ER Ca 2+ loss through an upregulation of Ca 2+ channels involved in unstimulated Ca 2+ influx. Assessment of the role of TRPM8, which was also positively correlated with NCS-1, may be challenging given the absence of TRPM8 in many commonly used breast cancer cell lines (Yapa et al., 2018). Alternatively, the positive association between ORAI1 and NCS-1 may be due to their association with breast cancers of the basal molecular subtype (Azimi et al., 2019). Likewise, lower levels of ORAI3 would indeed be predicted in the breast cancer cells with high levels of NCS-1 since ORAI3 levels are found to be lower in basal breast cancers (Azimi et al., 2019), which we have shown to have higher levels of NCS-1.
We also assessed the consequences of silencing NCS-1 on the effect of doxorubicin, a chemotherapeutic agent used in the treatment of TNBC (Gadi and Davidson, 2017), and whether ORAI1 silencing could phenocopy any effects of NCS-1 silencing. We identified that both NCS-1 and ORAI1 silencing enhanced the cell death induced by doxorubicin (1 µM) as determined using PI staining. We further showed that NCS-1 and ORAI1 silencing did not promote doxorubicin-induced PARP cleavage. These combined observations suggest that the mode of increased cell death is likely to be necrosis and not apoptosis. We hypothesize that the enhanced doxorubicin-induced cell death with NCS-1 silencing is mediated through its regulation of unstimulated basal Ca 2+ influx. This is supported by our observation that silencing of ORAI1, which mediates basal Ca 2+ influx in a variety of cells (Brandman et al., 2007;Ross et al., 2013;Subedi et al., 2018), also phenocopied the effects of NCS-1 silencing on doxorubicin-induced cell death. Indeed, suppression of basal Ca 2+ influx increases the effect of other cancer agents, as seen with the promotion of tamoxifen-induced cell death with TRPV6 silencing in T47D breast cancer cells (Bolanz et al., 2008;Peters et al., 2012).

Conclusion
This work defines a clear association between NCS-1 and the basal breast cancer molecular subtype, a subtype with poor prognosis. Our study is the first to identify an association between NCS-1 and unstimulated basal Ca 2+ influx in breast cancer cells and comprehensively characterize the role of NCS-1 in Ca 2+ homeostasis in breast cancer cells. NCS-1 silencing also enhanced cell death induced by doxorubicin treatment in MDA-MB-231 breast cancer cells. Further studies characterizing the role of NCS-1 in specific intracellular Ca 2+ and survival signaling pathways and in other basal breast cancer cell lines are now warranted. These findings will provide new insights into the potential role of NCS-1 as a modulator of therapeutic responses in basal breast cancers.