Role of SOCE architects STIM and Orai proteins in Cell Death

Calcium (Ca²⁺) signaling plays a critical role in regulating plethora of cellular functions including cell survival, proliferation and migration. The perturbations in cellular Ca²⁺ homeostasis can lead to cell death either by activating autophagic pathways or through induction of apoptosis. Endoplasmic reticulum (ER) is the major storehouse of Ca²⁺ within cells and a number of physiological agonists mediate ER Ca²⁺ release by activating IP₃ receptors (IP₃R). This decrease in ER Ca²⁺ levels is sensed by STIM, which physically interacts and activates plasma membrane Ca²⁺ selective Orai channels. Emerging literature implicates a key role for STIM1, STIM2, Orai1 and Orai3 in regulating both cell survival and death pathways. In this review, we will retrospect the work highlighting the role of STIM and Orai homologs in regulating cell death signaling. We will further discuss the rationales that could explain the dual role of STIM and Orai proteins in regulating cell fate decisions.     

ER Ca2+ release and store-operated Ca2+ entry – partners in crime or independent actors in oncogenic transformation?

Ca²⁺ is a pleiotropic messenger that controls life and death decisions from fertilisation until death. Cellular Ca²⁺ handling mechanisms show plasticity and are remodelled throughout life to meet the changing needs of the cell. In turn, as the demands on a cell alter, for example through a change in its niche environment or its functional requirements, Ca²⁺ handling systems may be targeted to sustain the remodelled cellular state. Nowhere is this more apparent than in cancer. Oncogenic transformation is a multi-stage process during which normal cells become progressively differentiated towards a cancerous state that is principally associated with enhanced proliferation and avoidance of death. Ca²⁺ signalling is intimately involved in almost all aspects of the life of a transformed cell and alterations in Ca²⁺ handling have been observed in cancer. Moreover, this remodelling of Ca²⁺ signalling pathways is also required in some cases to sustain the transformed phenotype. As such, Ca²⁺ handling is hijacked by oncogenic processes to deliver and maintain the transformed phenotype. Central to generation of intracellular Ca²⁺ signals is the release of Ca²⁺ from the endoplasmic reticulum intracellular (ER) Ca²⁺ store via inositol 1,4,5-trisphosphate receptors (InsP₃Rs). Upon depletion of ER Ca²⁺, store-operated Ca²⁺ entry (SOCE) across the plasma membrane occurs via STIM-gated Orai channels. SOCE serves to both replenish stores but also sustain Ca²⁺ signalling events. Here, we will discuss the role and regulation of these two signalling pathways and their interplay in oncogenic transformation.     

Regulation of CRAC channels by protein interactions and post-translational modification

Store-operated Ca²⁺ entry (SOCE) is a widespread mechanism to elevate the intracellular Ca²⁺ concentrations and stimulate downstream signaling pathways affecting proliferation, secretion, differentiation and death in different cell types. In immune cells, immune receptor stimulation induces intracellular Ca²⁺ store depletion that subsequently activates Ca²⁺-release-activated-Ca²⁺ (CRAC) channels, a prototype of store-operated Ca²⁺ (SOC) channels. CRAC channel opening leads to activation of diverse downstream signaling pathways affecting proliferation, differentiation, cytokine production and cell death. Recent identification of STIM1 as the endoplasmic reticulum Ca²⁺ sensor and Orai1 as the pore subunit of CRAC channels has provided the much-needed molecular tools to dissect the mechanism of activation and regulation of CRAC channels. In this review, we discuss the recent advances in understanding the associating partners and posttranslational modifications of Orai1 and STIM1 proteins that regulate diverse aspects of CRAC channel function.     

Assessment of cytosolic free calcium changes during ceramide-induced cell death in MDA-MB-231 breast cancer cells expressing the calcium sensor GCaMP6m

Alterations in Ca²⁺ signaling can regulate key cancer hallmarks such as proliferation, invasiveness and resistance to cell death. Changes in the regulation of intracellular Ca²⁺ and specific components of Ca²⁺ influx are a feature of several cancers and/or cancer subtypes, including the basal-like breast cancer subtype, which has a poor prognosis. The development of genetically encoded calcium indicators, such as GCaMP6, represents an opportunity to measure changes in intracellular free Ca²⁺ during processes relevant to breast cancer progression that occur over long periods (e.g. hours), such as cell death. This study describes the development of a MDA-MB-231 breast cancer cell line stably expressing GCaMP6m. The cell line retained the key features of this aggressive basal-like breast cancer cell line. Using this model, we defined alterations in relative cytosolic free Ca²⁺ ([Ca²⁺]_CYT) when the cells were treated with C2-ceramide. Cell death was measured simultaneously via assessment of propidium iodide permeability. Treatment with ceramide produced delayed and heterogeneous sustained increases in [Ca²⁺]_CYT. Where cell death occurred, [Ca²⁺]_CYT increases preceded cell death. The sustained increases in [Ca²⁺]_CYT were not related to the rapid morphological changes induced by ceramide. Silencing of the plasma membrane Ca²⁺ ATPase isoform 1 (PMCA1) was associated with an augmentation in ceramide-induced increases in [Ca²⁺]_CYT and also cell death. This work demonstrates the utility of GCaMP6 Ca²⁺ indicators for investigating [Ca²⁺]_CYT changes in breast cancer cells during events relevant to tumor progression, which occur over hours rather than minutes.     

Role of the calcium toolkit in cancer stem cells

Cancer stem cells are a subpopulation of tumor cells that proliferate, self-renew and produce more differentiated tumoral cells building-up the tumor. Responsible for the sustained growth of malignant tumors, cancer stem cells are proposed to play significant roles in cancer resistance to standard treatment and in tumor recurrence. Among the mechanisms dysregulated in neoplasms, those related to Ca²⁺ play significant roles in various aspects of cancers. Ca²⁺ is a ubiquitous second messenger whose fluctuations of its intracellular concentrations are tightly controlled by channels, pumps, exchangers and Ca²⁺ binding proteins. These components support the genesis of Ca²⁺ signals with specific spatio-temporal characteristics that define the cell response. Being involved in the coupling of extracellular events with intracellular responses, the Ca²⁺ toolkit is often hijacked by cancer cells to promote notably their proliferation and invasion. Growing evidence obtained during the last decade pointed to a role of Ca²⁺ handling and mishandling in cancer stem cells. In this review, after a general overview of the concept of cancer stem cells we analyse and discuss the studies and current knowledge regarding the complex roles of Ca²⁺ toolkit and signaling in these cells. We highlight that numbers of Ca²⁺ signaling actors promote cancer stem cell state and are associated with cell resistance to current cancer treatments and thus may represent promising targets for potential clinical applications.     

Calcium,mitochondria and cell metabolism: A functional triangle in bioenergetics

The versatility of mitochondrial metabolism and its fine adjustments to specific physiological or pathological conditions regulate fundamental cell pathways, ranging from proliferation to apoptosis. In particular, Ca²⁺ signalling has emerged as a key player exploited by mitochondria to tune their activity according with cell demand. The functional interaction between mitochondria and endoplasmic reticulum (ER) deeply impacts on the correct mitochondrial Ca²⁺ signal, thus modulating cell bioenergetics and functionality. Indeed, Ca²⁺ released by the ER is taken up by mitochondria where, both in the intermembrane space and in the matrix, it regulates the activity of transporters, enzymes and proteins involved in organelles' metabolism. In this review, we will briefly summarize Ca²⁺-dependent mechanisms involved in the regulation of mitochondrial activity. Moreover, we will discuss some recent reports, in which alterations in mitochondrial Ca²⁺ signalling have been associated with specific pathological conditions, such as neurodegeneration and cancer.     

Down‐regulation of Orai3 arrests cell‐cycle progression and induces apoptosis in breast cancer cells but not in normal breast epithelial cells

Breast cancer (BC) is the leading cancer in the world in terms of incidence and mortality in women. However, the mechanism by which BC develops remains largely unknown. The increase in cytosolic free Ca²⁺ can result in different physiological changes including cell growth and death. Orai isoforms are highly Ca²⁺ selective channels. In the present study, we analyzed Orai3 expression in normal and cancerous breast tissue samples, and its role in MCF‐7 BC and normal MCF‐10A mammary epithelial cell lines. We found that the expression of Orai3 mRNAs was higher in BC tissues and MCF‐7 cells than in normal tissues and MCF‐10A cells. Down‐regulation of Orai3 by siRNA inhibited MCF‐7 cell proliferation and arrested cell cycle at G1 phase. This phenomenon is associated with a reduction in CDKs 4/2 (cyclin‐dependent kinases) and cyclins E and D1 expression and an accumulation of p21^Waf1/Cip1 (a cyclin‐dependent kinase inhibitor) and p53 (a tumor‐suppressing protein). Orai3 was also involved in MCF‐7 cell survival. Furthermore, Orai3 mediated Ca²⁺ entry and contributed to intracellular calcium concentration ([Ca²⁺]_i). In MCF‐10A cells, silencing Orai3 failed to modify [Ca²⁺]_i, cell proliferation, cell‐cycle progression, cyclins (D1, E), CDKs (4, 2), and p21^Waf1/Cip1 expression. Our results provide strong evidence for a significant effect of Orai3 on BC cell growth in vitro and show that this effect is associated with the induction of cell cycle and apoptosis resistance. Our study highlights a possible role of Orai3 as therapeutic target in BC therapy. J. Cell. Physiol. 226: 542–551, 2011. © 2010 Wiley‐Liss, Inc.     

Crosstalk between calcium and reactive oxygen species signaling in cancer

The interplay between Ca²⁺ and reactive oxygen species (ROS) signaling pathways is well established, with reciprocal regulation occurring at a number of subcellular locations. Many Ca²⁺ channels at the cell surface and intracellular organelles, including the endoplasmic reticulum and mitochondria are regulated by redox modifications. In turn, Ca²⁺ signaling can influence the cellular generation of ROS, from sources such as NADPH oxidases and mitochondria. This relationship has been explored in great depth during the process of apoptosis, where surges of Ca²⁺ and ROS are important mediators of cell death. More recently, coordinated and localized Ca²⁺ and ROS transients appear to play a major role in a vast variety of pro-survival signaling pathways that may be crucial for both physiological and pathophysiological functions. While much work is required to firmly establish this Ca²⁺-ROS relationship in cancer, existing evidence from other disease models suggests this crosstalk is likely of significant importance in tumorigenesis. In this review, we describe the regulation of Ca²⁺ channels and transporters by oxidants and discuss the potential consequences of the ROS-Ca²⁺ interplay in tumor cells.     

MS4A15 drives ferroptosis resistance through calcium-restricted lipid remodeling

Ferroptosis is an iron-dependent form of cell death driven by biochemical processes that promote oxidation within the lipid compartment. Calcium (Ca²⁺) is a signaling molecule in diverse cellular processes such as migration, neurotransmission, and cell death. Here, we uncover a crucial link between ferroptosis and Ca²⁺ through the identification of the novel tetraspanin MS4A15. MS4A15 localizes to the endoplasmic reticulum, where it blocks ferroptosis by depleting luminal Ca²⁺ stores and reprogramming membrane phospholipids to ferroptosis-resistant species. Specifically, prolonged Ca²⁺ depletion inhibits lipid elongation and desaturation, driving lipid droplet dispersion and formation of shorter, more saturated ether lipids that protect phospholipids from ferroptotic reactive species. We further demonstrate that increasing luminal Ca²⁺ levels can preferentially sensitize refractory cancer cell lines. In summary, MS4A15 regulation of anti-ferroptotic lipid reservoirs provides a key resistance mechanism that is distinct from antioxidant and lipid detoxification pathways. Manipulating Ca²⁺ homeostasis offers a compelling strategy to balance cellular lipids and cell survival in ferroptosis-associated diseases.Subject terms: Lipidomics, Cancer genetics, Cell biology     

20-O-β-d-glucopyranosyl-20(S)-protopanaxadiol,a metabolite of ginseng,inhibits colon cancer growth by targeting TRPC channel-mediated calcium influx

Abnormal regulation of Ca²⁺ mediates tumorigenesis and Ca²⁺ channels are reportedly deregulated in cancers, indicating that regulating Ca²⁺ signaling in cancer cells is considered as a promising strategy to treat cancer. However, little is known regarding the mechanism by which Ca²⁺ affects cancer cell death. Here, we show that 20-O-β-d-glucopyranosyl-20(S)-protopanaxadiol (20-GPPD), a metabolite of ginseng saponin, causes apoptosis of colon cancer cells through the induction of cytoplasmic Ca²⁺. 20-GPPD decreased cell viability, increased annexin V-positive early apoptosis and induced sub-G1 accumulation and nuclear condensation of CT-26 murine colon cancer cells. Although 20-GPPD-induced activation of AMP-activated protein kinase (AMPK) played a key role in the apoptotic death of CT-26 cells, LKB1, a well-known upstream kinase of AMPK, was not involved in this activation. To identify the upstream target of 20-GPPD for activating AMPK, we examined the effect of Ca²⁺ on apoptosis of CT-26 cells. A calcium chelator recovered 20-GPPD-induced AMPK phosphorylation and CT-26 cell death. Confocal microscopy showed that 20-GPPD increased Ca²⁺ entry into CT-26 cells, whereas a transient receptor potential canonical (TRPC) blocker suppressed Ca²⁺ entry. When cells were treated with a TRPC blocker plus an endoplasmic reticulum (ER) calcium blocker, 20-GPPD-induced calcium influx was completely inhibited, suggesting that the ER calcium store, as well as TRPC, was involved. In vivo mouse CT-26 allografts showed that 20-GPPD significantly suppressed tumor growth, volume and weight in a dose-dependent manner. Collectively, 20-GPPD exerts potent anticarcinogenic effects on colon carcinogenesis by increasing Ca²⁺ influx, mainly through TRPC channels, and by targeting AMPK.     

Coupling endoplasmic reticulum stress to the cell death program in mouse melanoma cells: effect of curcumin

The microenvironment of cancerous cells includes endoplasmic reticulum (ER) stress the resistance to which is required for the survival and growth of tumors. Acute ER stress triggers the induction of a family of ER stress proteins that promotes survival and/or growth of the cancer cells, and also confers resistance to radiation and chemotherapy. Prolonged or severe ER stress, however, may ultimately overwhelm the cellular protective mechanisms, triggering cell death through specific programmed cell death (pcd) pathways. Thus, downregulation of the protective stress proteins may offer a new therapeutic approach to cancer treatment. In this regard, recent reports have demonstrated the roles of the phytochemical curcumin in the inhibition of proteasomal activity and triggering the accumulation of cytosolic Ca²⁺ by inhibiting the Ca²⁺-ATPase pump, both of which enhance ER stress. Using a mouse melanoma cell line, we investigated the possibility that curcumin may trigger ER stress leading to programmed cell death. Our studies demonstrate that curcumin triggers ER stress and the activation of specific cell death pathways that feature caspase cleavage and activation, p23 cleavage, and downregulation of the anti-apoptotic Mcl-1 protein.     

Remodelling of Ca2+ transport in cancer: how it contributes to cancer hallmarks?

Cancer involves defects in the mechanisms underlying cell proliferation, death and migration. Calcium ions are central to these phenomena, serving as major signalling agents with spatial localization, magnitude and temporal characteristics of calcium signals ultimately determining cell''s fate. Cellular Ca²⁺ signalling is determined by the concerted action of a molecular Ca²⁺-handling toolkit which includes: active energy-dependent Ca²⁺ transporters, Ca²⁺-permeable ion channels, Ca²⁺-binding and storage proteins, Ca²⁺-dependent effectors. In cancer, because of mutations, aberrant expression, regulation and/or subcellular targeting of Ca²⁺-handling/transport protein(s) normal relationships among extracellular, cytosolic, endoplasmic reticulum and mitochondrial Ca²⁺ concentrations or spatio-temporal patterns of Ca²⁺ signalling become distorted. This causes deregulation of Ca²⁺-dependent effectors that control signalling pathways determining cell''s behaviour in a way to promote pathophysiological cancer hallmarks such as enhanced proliferation, survival and invasion. Despite the progress in our understanding of Ca²⁺ homeostasis remodelling in cancer cells as well as in identification of the key Ca²⁺-transport molecules promoting certain malignant phenotypes, there is still a lot of work to be done to transform fundamental findings and concepts into new Ca²⁺ transport-targeting tools for cancer diagnosis and treatment.     

TRP channels in cell survival and cell death in normal and transformed cells

TRP channels form a superfamily of channel proteins exhibiting versatile regulatory characteristics with many channels participating in the regulation of Ca²⁺ homeostasis and influencing the cell fate. Multitude of evidence is emerging that the colocalization of TRP channels with Ca²⁺-sensing elements of specific regulatory pathways leading to either proliferation or apoptosis is what makes these channels participate in cell fate regulation and, in turn, determines the final effect of Ca²⁺ entry via the particular channel. This review focuses on the aspects of TRP channel localization and function that affect the balance between cell survival and death and how various dysregulations of these channels may lead to perturbed balance and onset of cancer.     

Roles of transient receptor potential canonical (TRPC) channels and reverse-mode Na/Ca exchanger on cell proliferation in human cardiac fibroblasts: Effects of transforming growth factor β1

Expression of transient receptor potential canonical channels (TRPC) and the effects of transforming growth factor-β1 (TGF-β1) on Ca²⁺ signals and fibroblast proliferation were investigated in human cardiac fibroblasts. The conventional and quantitative real-time RT-PCR, western blot, immunocytochemical analysis, and intracellular Ca²⁺ concentration [Ca²⁺]_i measurement were applied. Cell proliferation and cell cycle progression were assessed using MTT assays and fluorescence activated cell sorting. Human cardiac fibroblasts have the expression of TRPC1,3,4,6 mRNA and proteins. 1-oleoyl-2-acetyl-sn-glycerol (OAG) and thapsigargin induced extracellular Ca²⁺-mediated [Ca²⁺]_i rise. siRNA for knock down of TRPC6 reduced OAG-induced Ca²⁺ entry. Hyperforin as well as angiotensin II (Ang II) induced Ca²⁺ entry. KB-R7943, a reverse-mode Na⁺/Ca²⁺ exchanger (NCX) inhibitor, and/or replacement of Na⁺ with NMDG⁺ inhibited thapsigargin-, OAG- and Ang II-induced Ca²⁺ entry. Treatment with TGF-β1 increased thapsigargin-, OAG- and Ang II-induced Ca²⁺ entry with an enhancement of TRPC1,6 protein expression, suppressed by KB-R7943. TGF-β1 and AngII promoted cell cycle progression from G0/G1 to S/G2/M and cell proliferation. A decrease of the extracellular Ca²⁺ and KB-R7943 suppressed it. Human cardiac fibroblasts contain several TRPC-mediated Ca²⁺ influx pathways, which activate the reverse-mode NCX. TGF-β1 enhances the Ca²⁺ influx pathways requiring Ca²⁺ signals for its effect on fibroblast proliferation.     

Effect of thapsigargin on Ca2+ fluxes and viability in human prostate cancer cells

Effect of the carcinogen thapsigargin on human prostate cancer cells is unclear. This study examined if thapsigargin altered basal [Ca²⁺]_i levels in suspended PC3 human prostate cancer cells by using fura-2 as a Ca²⁺-sensitive fluorescent probe. Thapsigargin at concentrations between 10?nM and 10 µM increased [Ca²⁺]_i in a concentration-dependent fashion. The Ca²⁺ signal was reduced partly by removing extracellular Ca²⁺ indicating that Ca²⁺ entry and release both contributed to the [Ca²⁺]_i rise. This Ca²⁺ influx was inhibited by suppression of phospholipase A2, but not by inhibition of store-operated Ca²⁺ channels or by modulation of protein kinase C activity. In Ca²⁺-free medium, pretreatment with the endoplasmic reticulum Ca²⁺ pump inhibitor 2,5-di-(t-butyl)-1,4-hydroquinone (BHQ) nearly abolished thapsigargin-induced Ca²⁺ release. Conversely, pretreatment with thapsigargin greatly reduced BHQ-induced [Ca²⁺]_i rise, suggesting that thapsigargin released Ca²⁺ from the endoplasmic reticulum. Inhibition of phospholipase C did not change thapsigargin-induced [Ca²⁺]_i rise. At concentrations of 1-10 µM, thapsigargin induced cell death that was partly reversed by chelation of Ca²⁺ with BAPTA/AM. Annexin V/propidium iodide staining data suggest that apoptosis was partly responsible for thapsigargin-induced cell death. Together, in PC3 human prostate cancer cells, thapsigargin induced [Ca²⁺]_i rises by causing phospholipase C-independent Ca²⁺ release from the endoplasmic reticulum and Ca²⁺ influx via phospholipase A2-sensitive Ca²⁺ channels. Thapsigargin also induced cell death via Ca²⁺-dependent pathways and Ca²⁺-independent apoptotic pathways.     

Calcium signaling and the therapeutic targeting of cancer cells

The calcium signal is implicated in a variety of processes important in tumor progression (e.g. proliferation and invasiveness). The calcium signal has also been shown to be important in other processes important in cancer progression including the development of resistance to current cancer therapies. In this review, we discuss how Ca²⁺ channels, pumps and exchangers may be drug targets in some cancer types. We consider what factors should be taken into account when considering an optimal Ca²⁺ channel, pump or exchanger as a candidate for further assessment as a novel drug target in cancer. We also present and summarize how some therapies for the treatment of cancer intersect with Ca²⁺ signaling and how pharmacological manipulation of the machinery of Ca²⁺ signaling could promote the effectiveness of some therapies. We also review new therapeutic opportunities for Ca²⁺ signal modulators in the context of the tumor microenvironment.     

Oncogenic KRAS suppresses store-operated Ca2+ entry and ICRAC through ERK pathway-dependent remodelling of STIM expression in colorectal cancer cell lines

The KRAS GTPase plays a fundamental role in transducing signals from plasma membrane growth factor receptors to downstream signalling pathways controlling cell proliferation, survival and migration. Activating KRAS mutations are found in 20% of all cancers and in up to 40% of colorectal cancers, where they contribute to dysregulation of cell processes underlying oncogenic transformation. Multiple KRAS-regulated cell functions are also influenced by changes in intracellular Ca²⁺ levels that are concurrently modified by receptor signalling pathways. Suppression of intracellular Ca²⁺ release mechanisms can confer a survival advantage in cancer cells, and changes in Ca²⁺ entry across the plasma membrane modulate cell migration and proliferation. However, inconsistent remodelling of Ca²⁺ influx and its signalling role has been reported in studies of transformed cells. To isolate the interaction between altered Ca²⁺ handling and mutated KRAS in colorectal cancer, we have previously employed isogenic cell line pairs, differing by the presence of an oncogenic KRAS allele (encoding KRAS^G13D), and have shown that reduced Ca²⁺ release from the ER and mitochondrial Ca²⁺ uptake contributes to the survival advantage conferred by oncogenic KRAS. Here we show in the same cell lines, that Store-Operated Ca²⁺ Entry (SOCE) and its underlying current, I_CRAC are under the influence of KRAS^G13D. Specifically, deletion of the oncogenic KRAS allele resulted in enhanced STIM1 expression and greater Ca²⁺ influx. Consistent with the role of KRAS in the activation of the ERK pathway, MEK inhibition in cells with KRAS^G13D resulted in increased STIM1 expression. Further, ectopic expression of STIM1 in HCT 116 cells (which express KRAS^G13D) rescued SOCE, demonstrating a fundamental role of STIM1 in suppression of Ca²⁺ entry downstream of KRAS^G13D. These results add to the understanding of how ERK controls cancer cell physiology and highlight STIM1 as an important biomarker in cancerogenesis.