Sodium regulates PLC and IP3R‐mediated calcium signaling in invasive breast cancer cells

Abstract Intracellular Ca2+ signaling and Na+ homeostasis are inextricably linked via ion channels and co‐transporters, with alterations in the concentration of one ion having profound effects on the other. Evidence indicates that intracellular Na+ concentration ([Na+]i) is elevated in breast tumors, and that aberrant Ca2+ signaling regulates numerous key cancer hallmark processes. The present study therefore aimed to determine the effects of Na+ depletion on intracellular Ca2+ handling in metastatic breast cancer cell lines. The relationship between Na+ and Ca2+ was probed using fura‐2 and SBFI fluorescence imaging and replacement of extracellular Na+ with equimolar N‐methyl‐D‐glucamine (0Na+/NMDG) or choline chloride (0Na+/ChoCl). In triple‐negative MDA‐MB‐231 and MDA‐MB‐468 cells and Her2+ SKBR3 cells, but not ER+ MCF‐7 cells, 0Na+/NMDG and 0Na+/ChoCl resulted in a slow, sustained depletion in [Na+]i that was accompanied by a rapid and sustained increase in intracellular Ca2+ concentration ([Ca2+]i). Application of La3+ in nominal Ca2+‐free conditions had no effect on this response, ruling out reverse‐mode NCX activity and Ca2+ entry channels. Moreover, the Na+‐linked [Ca2+]i increase was independent of membrane potential hyperpolarization (NS‐1619), but was inhibited by pharmacological blockade of IP3 receptors (2‐APB), phospholipase C (PLC, U73122) or following depletion of endoplasmic reticulum Ca2+ stores (cyclopiazonic acid). Thus, Na+ is linked to PLC/IP3‐mediated activation of endoplasmic reticulum Ca2+ release in metastatic breast cancer cells and this may have an important role in breast tumors where [Na+]i is perturbed.


| INTRODUCTION
Early detection has played an important role for timely clinical intervention in breast cancer; however, development of novel therapies which selectively kill malignant cells remains a central research goal. Key hallmarks of cancer include unlimited replicative capacity, self-sufficiency of growth, apoptosis resistance, angiogenesis, and ultimately tissue invasion (Hanahan & Weinberg, 2016). Importantly, intracellular ion signaling pathways (in particular Ca 2+ and Na + signaling) play a key role in regulating many of these hallmark processes in breast cancer (Bruce & James, 2020;Capatina et al., 2022;Leslie et al., 2019;Yang & Brackenbury, 2022), and thus ion signaling may be a novel therapeutic locus.
Intracellular [Na + ] ([Na + ] i ) and intracellular [Ca 2+ ] ([Ca 2+ ] i ) signaling is achieved via a multiplicity of ion channels and transporters located both at the plasma membrane and on intracellular organelles. These channels harness the electrochemical gradient of Na + and Ca 2+ across cell membranes to achieve their function, with [Na + ] i and [Ca 2+ ] i typically tightly regulated by various ion transporters (Bruce, 2018;Leslie et al., 2019). Importantly, Ca 2+ and Na + signaling are inextricably linked via mechanisms mutually regulated by both ions, such as the plasma membrane Na + /Ca 2+ exchanger (NCX; which is responsible for bulk export of Ca 2+ from the cytosol) and the mitochondrial Na + /Ca 2+ /Li + exchanger (which regulates mitochondrial Ca 2+ and Na + and thus bioenergetics, Hernansanz-Agustín et al., 2020). Thus, changes in how the cell handles one ion would be expected to impact upon the other. Moreover, the interplay between [Na + ] i and [Ca 2+ ] i handling has important implications in breast cancer since [Na + ] i is elevated in malignant breast cancer cells, particularly in those exhibiting aberrant Na + channel expression (Cameron et al., 1980;James et al., 2022;Ouwerkerk et al., 2007;Yang et al., 2020).
The present study aimed to determine the effects of Na + depletion on intracellular Ca 2+ handling in metastatic breast cancer cell lines in order to probe the relationship between Na + and Ca 2+ in breast cancer. A commonly recognized method to probe the role of Na + transport in cell physiology is by replacement of extracellular Na + with equimolar N-methyl-D-glucamine (0Na + /NMDG) or choline chloride (0Na + /ChoCl). These replacement cations maintain the osmotic balance across the cell membrane, yet are not transported via Na + channels or transporters. Breast cancer cells can be subdivided into three main categories based on expression of the estrogen receptor (ER), progesterone receptor and human epidermal growth factor 2 (Her2). In triple-negative MDA-MB-231 and MDA-MB-468 cells (lacking all three receptors) and Her2+ SKBR3 cells, removal of Na + from the extracellular space led to a slow, sustained depletion in [Na + ] i that was accompanied by a rapid and sustained increase in intracellular [Ca 2+ ] i . Interestingly, this response was absent in ER+ MCF-7 cells. The observed [Ca 2+ ] i transient was not due to Ca 2+ entry from the extracellular space, ruling out reverse-mode NCX activity. Moreover, it was independent of membrane potential (V m ) and was inhibited by depletion of the endoplasmic reticulum Ca 2+ store or by pharmacological blockade of inositol (1,4,5) trisphosphate (IP 3 ) receptors (IP 3 Rs) or phospholipase C (PLC). These data reveal a previously unreported Na + -linked activation of endoplasmic reticulum Ca 2+ release in metastatic breast cancer cells, which may have an important role in breast tumors where [Na + ] i is elevated or perturbed. Moreover, the dramatic Ca 2+ release observed upon Na + depletion serves as a cautionary note for those utilizing similar Na + replacement methods to study the role of Na + transport in cellular processes. MCF-7 cells were gifts from M. Djamgoz (Imperial College London) and SKBR3 cells were a gift from J. Rae (University of Michigan). Molecular identity of MDA-MB-231, MCF-7, and SKBR3 cells was verified by short tandem repeat analysis (Masters et al., 2001). MDA-MB-468 cells were obtained for this study directly from the American Type Culture Collection (ATCC).

| Drug preparation
Stocks of NS-1619, 2-APB, dantrolene, cyclopiazonic acid (CPA), and ionomycin were prepared in DMSO. The final DMSO concentration in working solutions was 0.1%. Stocks of La 3+ were prepared using ultrapure water. Drugs were diluted into HEPES-PSS immediately prior to use.

| Measurement of [Ca 2+ ] i responses
Changes in [Ca 2+ ] i in response to treatment were measured as either the maximum fura-2 ratio change (ΔR max ) or the area under the curve (AUC). The fura-2 fluorescence ratios for each trace were first normalized to the ratio at time 0 (R/R 0 ) to control for variability in starting ratio between cells. To obtain a pretreatment baseline, the average ratio across the six timepoints (30 seconds at an acquisition interval of 5 seconds) immediately prior to treatment application was determined. ΔR max was defined as the maximum increase from this baseline during the treatment period. The AUC was defined as the area under the curve during the entire treatment period (i.e., 10 min) in fura-2 ratio unit seconds (R.s). Where no [Ca 2+ ] i response was elicited in response to treatment, cells were stimulated with ionomycin (3 μM) to ensure a change in [Ca 2+ ] i could be measured. Cells in which no fura-2 fluorescence change was observed were excluded from analysis.

| Data analysis
Data analysis and statistical comparisons were performed using Microsoft Excel and GraphPad Prism 9. Statistical comparisons were performed using a nested t-test for side-by-side comparisons and a nested one-way ANOVA with posthoc Tukey test for multiple comparisons. Within each figure, nested data are presented from individual experiments (3-6 independent repeats, each containing 12-40 independent cells) and the individual experimental means ± S.E.M.

| Na + depletion drives an increase in [Ca 2+ ] i in breast cancer cells
Metastatic breast cancer cells exhibit elevated [Na + ] i (Leslie et al., 2019) that may regulate reverse-mode (Ca 2+entry) NCX activity (Pappalardo et al., 2014;Verkhratsky et al., 2018) in these cells. To assess the effects of lowering extracellular Na + on [Ca 2+ ] i , MDA-MB-231 cells were loaded with fura-2 AM (4 μM) and perfused with sequential pulses of HEPES-PSS where Na + had been replaced with either equimolar N-methyl D glucamine (0Na + / NMDG) or equimolar choline chloride (0Na + /ChoCl) to remove extracellular Na + while maintaining osmotic balance. Fura-2 fluorescence imaging revealed that, using either maneuver, removal of extracellular Na + resulted in a steep increase in [Ca 2+ ] i that returned to baseline upon reperfusion with standard Na + -containing HEPES-PSS (Figure 1ai,bi Figure S1A). Interestingly, perfusion of 0Na + /NMDG had no effect on [Ca 2+ ] i in the ER+ breast cancer cell line MCF-7 (Figure 1c), but induced a [Ca 2+ ] i transient in the Her2+ breast cancer cell line SKBR3 ( Figure 1d); nevertheless, both MCF-7 and SKBR3 cells exhibited depletion of [Na + ] i upon perfusion with 0Na + /NMDG (Supplementary Figure S1b,c). Taken together, these data suggest that the [Ca 2+ ] i rise upon removal of extracellular Na + and concomitant depletion of [Na + ] i is present only in certain breast cancer cell lines.

| The [Ca 2+ ] i increase induced by
Na + -depletion is not due to Ca 2+ influx from the extracellular space We reasoned that the increase in [Ca 2+ ] i observed under 0Na + conditions likely originated from one of two compartments: Ca 2+ release from intracellular stores (most likely the endoplasmic reticulum) or Ca 2+ influx from the extracellular space. To determine whether Ca 2+ influx from the extracellular space contributed to the [Ca 2+ ] i responses observed, MDA-MB-231 cells were perfused with 0Na + /NMDG in nominal Ca 2+ -free conditions with La 3+ (1 mM); La 3+ in the millimolar range is a known inhibitor of both Ca 2+ entry from and Ca 2+ efflux to the extracellular space (James et al., 2013). La 3+ was applied for 5 minutes prior to application of 0Na + /NMDG (Figure 2b). Compared with control cells (Figure 2a), La 3+ had no effect on the [Ca 2+ ] i transients elicited following 0Na + / NMDG application, as measured by either the maximum change in fura-2 ratio (ΔR max , Figure 2c) or the area under the curve (AUC, Figure 2d). These results indicate that Ca 2+ influx via agonist-induced and store-operated Ca 2+ entry mechanisms plays no role in the [Ca 2+ ] i transients induced following application of 0Na + /NMDG, and that the provenance of this Ca 2+ response must be some compartment other than the extracellular space.

| Elevations in [Ca 2+ ] i induced by
Na + -depletion are not due to changes in membrane potential Maneuvers that deplete extracellular Na + such as 0Na + / NMDG are known to cause V m hyperpolarization (Yang et al., 2020), thereby altering ion flux across the plasma membrane. Moreover, V m hyperpolarization would increase the driving force for Ca 2+ entry. To determine whether such changes in V m led to the [Ca 2+ ] i transient observed under 0Na + /NMDG conditions, we applied the large-conductance Ca 2+ -activated K + channel (K Ca 1.1) activator NS-1619 at a concentration (10 μM) that hyperpolarizes V m in MDA-MB-231 cells to a similar degree to 0Na + /NMDG (Yang et al., 2020). Similar to control cells (Figure 3a However, an increased concentration of NS-1619 (40 μM, Figure 3c) elicited an increase in [Ca 2+ ] i that was significantly greater than any change observed in either the control cells (p < 0.01 for both ΔR max and AUC, Figure 3e,f) or those treated with 10 μM NS-1619 (p < 0.01 for both ΔR max and AUC, Figure 3e,f), indicating that dramatic changes in V m can indeed alter [Ca 2+ ] i . Importantly, the effects of 40 μM NS-1619 on [Ca 2+ ] i were abolished following pretreatment with La 3+ (1 mM, p < 0.05 for both ΔR max and AUC, Figure 3c-f), indicating that the NS-1619 induced rise was due to Ca 2+ influx from the extracellular space ( Figure 3d). Given that Ca 2+ influx plays no role in the [Ca 2+ ] i transients observed following application of 0Na + / NMDG (Figure 2), these results suggest that neither Ca 2+ influx nor hyperpolarization of V m play a role in changes in [Ca 2+ ] i induced by application 0Na + /NMDG.

| Depletion of ER Ca 2+ stores inhibits the increase in [Ca 2+ ] i induced by 0Na + /NMDG
Having ruled out Ca 2+ influx as being responsible for the [Ca 2+ ] i transients induced following application of 0Na + / NMDG, we tested whether depletion of the intracellular Ca 2+ stores within the endoplasmic reticulum affected the observed rises in [Ca 2+ ] i . To deplete endoplasmic reticulum Ca 2+ stores, cells were treated with the sarcoplasmic/ endoplasmic reticulum Ca 2+ ATPase (SERCA) inhibitor cyclopiazonic acid (CPA, 30 μM) in nominal Ca 2+ -free conditions (to prevent store-operated Ca 2+ entry), resulting in a transient leak of Ca 2+ from the endoplasmic reticulum that was subsequently cleared from the cell (Figure 4b), presumably by the plasma membrane Ca 2+ ATPase (James et al., 2015). These experiments were performed without EGTA since it was observed that inclusion of EGTA (1 mM) within the Ca 2+ -free buffer inhibited the [Ca 2+ ] i increases elicited by 0Na + /NMDG (Supplementary Figure S2). Compared with control cells, (Figure 4a), pretreatment with CPA completely abolished the increase in [Ca 2+ ] i elicited by 0Na + /NMDG (Figure 4b-d; p < 0.01 for both ΔR max and AUC). These results indicate that the 0Na + /NMDGinduced [Ca 2+ ] i transient is likely due to release of Ca 2+ from the intracellular endoplasmic reticulum Ca 2+ stores.

| Na + depletion drives increases in [Ca 2+ ] i via a G-protein coupled receptor/IP 3 receptor-mediated mechanism
Given the evidence implicating endoplasmic reticulum Ca 2+ release in the changes in [Ca 2+ ] i observed upon application of 0Na + /NMDG, we next sought to determine the key molecular players responsible. Ca 2+ is classically released from the endoplasmic reticulum via inositol (1,4,5) triphosphate receptors (IP 3 Rs) and ryanodine receptors (RyRs; Berridge et al., 2000). To determine which of these release mechanisms was responsible for [Ca 2+ ] i transients F I G U R E 2 [Ca 2+ ] i transients induced by removal of extracellular Na + in breast cancer cells are not due to Ca 2+ entry. MDA-MB-231 cells were loaded with fura-2 AM (4 μM) and perfused with HEPES-PSS. Extracellular Na + was replaced with equimolar 0Na + /NMDG to maintain osmotic balance (a). To block all Ca 2+ entry from the extracellular space, cells were pretreated with 5 min of perfusion with Ca 2+free HEPES containing La 3+ (1 mM, b). Ionomycin (3 μM) was applied at the end of an experiment to elicit a [Ca + ] i increase as a positive control. The maximum change in fluorescence ratio (ΔR max , c) and area under the curve (AUC, d) were compared using a nested t-test for side-by-side comparisons; n = 4 independent experiments for each condition, ns, not significant compared with control. Data presented are nested values for individual cells grouped by experiment (gray dots) and experimental means ± SEM (black line and bars). upon application of 0Na + /NMDG, we perfused MDA-MB-231 cells with 0Na + /NMDG following pretreatment (10 min) with either the IP 3 R inhibitor 2-APB (50 μM) or the RyR inhibitor dantrolene (10 μM). Compared to control cells (Figure 5ai,bi), 2-APB significantly reduced the [Ca 2+ ] i transient induced by 0Na +/ NMDG (Figure 5aiiaiv; ΔR max , p < 0.05; AUC, p < 0.001), whereas dantrolene (10 μM, Figure 5bii) had no effect (Figure 5biii,biv). These data suggest that Na + depletion induces Ca 2+ release from the endoplasmic reticulum via IP 3 Rs.

| DISCUSSION
The present study is the first to describe a relationship in metastatic breast cancer cells between Na + depletion and a subsequent dramatic rise in [Ca 2+ ] i that is mediated by a PLC-and IP 3 R-dependent mechanism. These results provide further evidence of an interplay between Ca 2+ and Na + signaling in breast cancer cells that may have important implications for understanding cancer cell physiology. Through pharmacological maneuvers we rule out any role for reverse-mode NCX activity, V m and Ca 2+ entry F I G U R E 3 Hyperpolarization of the membrane potential (V m ) plays no role in [Ca 2+ ] i transients in breast cancer cells induced by removal of extracellular Na + . MDA-MB-231 cells were loaded with fura-2 AM (4 μM) and perfused with HEPES-PSS in the presence of the large-conductance Ca 2+ -activated K + channel (K Ca 1.1) activator NS-1619 to hyperpolarize V m . Representative traces are shown for cells perfused without NS-1619 (a) and with 10 μM NS-1619 (b) or 40 μM NS-1619 (c). Application of La 3+ (d) determined that [Ca 2+ ] i changes observed following treatment with 40 μM NS-1619 were due to Ca 2+ entry. Ionomycin (3 μM) was applied at the end of an experiment to elicit a [Ca 2+ ] i increase as a positive control. The maximum change in fluorescence ratio (ΔR max , e) and area under the curve (AUC, f) were compared using a nested one-way ANOVA with post-hoc Tukey test for multiple comparisons; n = 3-4 independent experiments for each condition. *p < 0.05; **p < 0.01 compared with control. Data presented are nested values for individual cells grouped by experiment (gray dots) and experimental means ± SEM (black line and bars). in this response. We also found that CPA abolished the [Ca 2+ ] i transients elicited by 0Na + /NMDG application; CPA is a well characterized SERCA blocker, providing further indirect evidence that this phenomenon was mediated by an IP 3 R-mediated endoplasmic reticulum Ca 2+ release mechanism. Furthermore, while [Ca 2+ ] i transients were observed upon Na + depletion in MDA-MB-231, MDA-MB-468, and SKBR3 cells, no such response was observed in MCF-7 cells. This finding indicates that the Na +dependent Ca 2+ increase is not a universal feature across all breast cancer cell lines. Further work is required to determine the underlying reason for the difference in Na + and Ca 2+ signaling between different cancer cell lines.
The present study establishes that Na + regulates a PLC/ IP 3 -dependent Ca 2+ release in metastatic breast cancer cells; however, the molecular identity of the Na + "sensor" that initiates this event remains unknown. Interestingly, Na + has been described as an endogenous regulator of Class A GPCRs (White et al., 2018) via allosteric regulation of agonist binding (Agasid et al., 2021;Katritch et al., 2014;Zarzycka et al., 2019). While we cannot presently rule out whether it is the depletion of cytosolic [Na + ] or removal of extracellular Na + that induces this [Ca 2+ ] i transient in the current study, these previous studies provide a strong potential candidate for the underlying trigger mechanism. Indeed, in these studies, Na + depletion appeared to increase basal GPCR activity in the absence of agonist, suggesting that Na + may act as a negative modulator of GPCR activation (Agasid et al., 2021;Katritch et al., 2014;Zarzycka et al., 2019). Interestingly, recently obtained X-ray structures of Class A GPCRs have identified a Na + binding site in the vicinity of the agonist binding site that is exposed to the extracellular space (Yuan et al., 2013;Katritch et al., 2014). Moreover, computer simulations have suggested Class A GPCRs exhibit a transient water-filled channel connecting this Na + binding site to the cytoplasm (Hu et al., 2019;Vickery et al., 2018;Yuan et al., 2014Yuan et al., , 2015. Na + translocation via such a channel would be expected to be electrogenic, and thus impact upon V m as well as Na + handling (Katritch et al., 2014;Shalaeva et al., 2019. This has important implications for cancer cells exhibiting altered Na + dynamics, since V m is an important regulator of cancer cell behavior (Yang & Brackenbury, 2022); moreover, the altered [Na + ] i observed in breast tumors relative to healthy tissue (James et al., 2022) might be expected to disrupt GPCR regulation by Na + .
It is worthy to note that, in the present study, the PLC inhibitor U73122 had a more potent inhibitory effect on the [Ca 2+ ] i rise than the IP 3 R inhibitor 2-APB; the reasons for this are not clear, and suggest that U73122 is a more effective inhibitor of this pathway than 2-APB. However, 2-APB has been shown to target other channels, including certain TRP channel subtypes (Singh et al., 2018), which may have contributed to altered cation concentrations during these F I G U R E 4 [Ca 2+ ] i transients induced by removal of extracellular Na + in breast cancer cells require ER Ca 2+ stores. MDA-MB-231 cells were loaded with fura-2 AM (4 μM) and Ca 2+ imaging performed during replacement of extracellular Na + with equimolar (0Na + / NMDG) in the presence or absence of the SERCA inhibitor cyclopiazonic acid (CPA, 30 μM). Representative traces are shown for cells perfused without CPA (a) or where cells were pretreated for 20 min CPA prior to application of 0Na + /NMDG (b). The maximum change in fluorescence ratio (ΔR max , c) and area under the curve (AUC, d) were compared using a nested t-test for side-by-side comparisons; n = 4-6 independent experiments for each condition; **p < 0.01 compared with control. Data presented are nested values for individual cells grouped by experiment (gray circles) and experimental means ± SEM (black line and bars).
experiments. These findings also present a cautionary tale for cell physiologists using the methods employed in this study for exploring the relationship between Na + conductance, Ca 2+ signaling and wider cell behavior. Application of 0Na + /NMDG or 0Na + /ChoCl is widely utilized for isolating cell responses from the influence of extracellular Na + ; however, these maneuvers are not inert (Thuma & Hooper, 2018). In addition to assessing for reverse mode NCX activity (Chovancova et al., 2020), Na + regulation of GPCRs (and resultant effects on Ca 2+ dynamics) should be taken into careful consideration and controlled for when using 0Na + /NMDG, 0Na + /ChoCl or similar maneuvers to probe Na + conductance mechanisms.
Interestingly, inclusion of EGTA in the Ca 2+ -free extracellular buffer abolished the [Ca 2+ ] i transients elicited by 0Na + /NMDG application, suggesting that EGTA exerts an inhibitory effect on the PLC and IP 3 -mediated Ca 2+ release upon Na + depletion. It has previously been shown that F I G U R E 5 [Ca 2+ ] i transients induced by removal of extracellular Na + require activation of IP 3 receptor and phospholipase C, but are independent of ryanodine receptor activation. MDA-MB-231 cells were loaded with fura-2 AM (4 μM) and Ca 2+ imaging performed during replacement of extracellular Na + with equimolar (0Na + /NMDG) in the absence or presence of the IP 3 receptor inhibitor 2-APB (50 μM, ai and aii, respectively), the ryanodine receptor inhibitor dantrolene (10 μM, bi and bii, respectively), and the phospholipase C inhibitor U73122 (2 μM, ci and cii, respectively). For each condition, cells were pretreated with drug for 10 min prior to application of 0Na + /NMDG. Ionomycin (3 μM) was applied at the end of an experiment to elicit a [Ca + ] i increase as a positive control. The maximum change in fluorescence ratio (ΔR max ; aiii, 2-APB; biii, dantrolene; ciii, U73122) and area under the curve (AUC; aiv, 2-APB; biv, dantrolene; civ, U73122) were compared using a nested t-test for side-by-side comparisons; n = 3-5 independent experiments for each condition; *p < 0.05; ***p < 0.001 compared with control. Data presented are nested values for individual cells grouped by experiment (gray dots) and experimental means ± SEM (black line and bars).
EGTA can inhibit mobilization of IP 3 -sensitive Ca 2+ stores (Combettes & Champeil, 1994;Finch & Goldin, 1994) in cerebellar microsomes. However, EGTA is considered membrane impermeable, and thus would not be expected to exert a direct effect on IP 3 Rs in the present study. This suggests that EGTA instead exerts its effects at the extracellular side of the plasma membrane via an at present undefined mechanism. Such a mechanism could be either by a direct effect on the putative GPCR involved or via chelation of extracellular divalent cations required for proper transmembrane protein function. Alternatively, it has been argued that extracellularly-applied EGTA may be taken up into cells via highly Ca 2+ -permeable cation channels (e.g., TRPA1, TRPV1, P2X7) and/or via fluid-phase endocytosis and subsequent release into the cytosol (Liu et al., 2020). Further work is required to resolve these possibilities.
Beyond the present study, very little is known about the link between Ca 2+ signaling and Na + homeostasis in the context of breast cancer. Evidence indicates that Ca 2+ oscillations in MDA-MB-231 cells are regulated by aberrantly expressed VGSCs (Rizaner et al., 2016). Moreover, elevations in extracellular Na + cause p-glycoproteininduced resistance to paclitaxel in breast cancer cells via Ca 2+ signaling (Babaer et al., 2018). Of particular relevance to the current study (where Na + depletion activated IP 3 R-dependent Ca 2+ signaling), IP 3 Rs are upregulated in breast cancer (Foulon et al., 2022), and estradiol-induced IP 3 R signaling regulates breast cancer cell proliferation (Szatkowski et al., 2010). Either pharmacological inhibition or knockdown of IP 3 Rs in breast cancer cells led to dysregulated bioenergetics, and autophagy, cell cycle arrest, and cell death (Mound et al., 2013;Singh et al., 2017). Thus, the emerging evidence for altered Na + and Ca 2+ handling and their inextricable link via common channels and transporters hint at a hitherto relatively underexplored avenue in the context of cancer. The present study provides further evidence of such a link between Ca 2+ and Na + signaling that may have important implications for regulating cancer cell behavior and future therapy.

AUTHOR CONTRIBUTIONS
The project was designed by WJB and ADJ; experiments were carried out by ADJ, NS, KU, IJ, and RS with the assistance of GJOE; data analysis was performed by ADJ, NS, KU, IJ, and RS; the manuscript was prepared by ADJ and WJB; ADJ, WJB, GJOE, and SC contributed to study design, interpretation of the data, and critical revision of the paper for important intellectual content.