Publié dans FEBS J. 2013;280(21):5500-10.
By Dubois Charlotte1, Vanden Abeele Fabien1, and Prevarskaya Natacha1
1
Inserm, U-1003, Equipe labellisée par la Ligue Nationale contre le cancer, Villeneuve d’Ascq, France, Laboratory of Excellence, Ion Channels Science and Therapeutics, Université des Sciences et Technologies de Lille (USTL), Villeneuve d’Ascq, France
Abstract
Calcium is a universal messenger regulating many physiological functions including this vital one : namely the ability of the cell to undergo orderly self-destruction upon completion of its mission, called apoptosis. In physiopathological conditions such as cancer, apoptotic processes become deregulated, leading to apoptosis-resistant phenotypes. Recently perturbations of cellular calcium homeostasis have been described in apoptosis-resistant cell phenotypes. Thereby, new molecular actors have been identified, offering more accurate research possibilities in the field of apoptosis resistance and providing the bases for more rational approaches to cancer treatments. In this review, we focus on the calcium-transporting protein-dependent pathways involved in apoptosis, which are deregulated by cancer. We present the calcium-transporting proteins involved in the deregulation of apoptosis and those chemotherapies which target actors in calcium-induced apoptosis.
Introduction
Cancer is caused by defects in the mechanisms underlying cell proliferation and cell death. The development of tumours results from excessive cell proliferation combined with inhibition of cell apoptosis that eventually leads to imbalances in tissue homeostasis and uncontrolled proliferation [1]. The molecular machineries of proliferation and apoptosis are different, with proliferation relying on cyclin-dependent protein kinases (CDKs) – regulators of cell division cycle [2] and apoptosis primarily dependent on caspases – cysteine proteases executing a cell death programme [3]. Nevertheless, calcium ions are central to both phenomena, serving as major signalling agents of calcium signals ultimately determining cell's fate. From these observations, it is clear that changes in cytosolic free calcium ([Ca2+]i) alone are insufficient for governing such diverse processes deciding cell fate. Therefore, the amplitude, spatial localization and temporal characteristics of calcium signals are of major importance in determining death, survival and
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proliferation.[4] [5]. The formation of local signalling complexes due to restricted calcium diffusion[6] and the implication of molecular actors (i.e calcium channels, associated proteins, ions exchangers…) may well allow even greater specialization of the cellular responses controlled by calcium. Well documented reviews describing the developpment of tumors, and other calcium dependent pathways such as migration [7] [8], differenciation [9] [10], and proliferation [11] [12] have ever been published by specialists in their domain. In-depth analysis of complex apoptosis machinery in physiological conditions have already been presented in a number of recent specialized reviews [13] [14] [15] [16] [17], therefore we restrict ourselves to outlining those actors, involved in the failure of Ca2+-dependent apoptosis observed in various cancer models.
Apoptosis is an orderly physiological process that allows the elimination of cells that have already completed, or for any reason are incapable of performing their physiological function and therefore are no longer necessary. It is important for normal embryonic development and for the maintenance of tissue homeostasis in the adult organism. Imbalance in apoptosis can cause diseases such as cancer. The calcium dependence of apoptosis has been well-defined and comprehensively described in numerous review articles [18][19][20]. The calcium apoptotic signalling involves two critical factors: sustained elevation of intracellular calcium concentration ([Ca2+]i) and a prolonged decrease in the calcium concentration of the endoplasmic reticulum ([Ca2+] ER), with a variable contribution of each factor depending on cell type. Thus, to effectively evade apoptosis, cancer cells must employ mechanisms that do two things. Firstly, they must substantially reduce or even prevent calcium influx by downregulating the expression of Ca2+-permeable channels or the signalling pathways that lead to their activation. Secondly, they must, enable them to adapt to the chronic underfilling of the ER Ca2+ store [21] [22]. Reduced Ca2+ influx in cancer cells prevents Ca2+ overload in response to pro-apoptotic stimuli, thereby impairing the effectiveness of mitochondrial and cytoplasmic apoptotic pathways, whereas adaptation to the reduced ER Ca2+ content diminishes activation of ER stress-dependent responses.
In this review, we discuss of the remodelling of calcium signalling actors involved in the defect of apoptosis observed during the progression of various cancer, and the potential therapies based on calcium pathways used either in the clinic or are currently under development.
I. Calcium-transporting protein-induced apoptosis deregulated during cancer A. The entry to the cell:
It is generally accepted that intracellular calcium signals destined to support life processes such as proliferation, differentiation, migration, secretion have a dynamic nature and are structured in space and time, often taking the form of calcium oscillations, waves, sparks, spikes, flickers, etc. [23] [24]. Calcium is toxic to the cell and for this reason the basal intracellular concentration of calcium is usually maintained at a very low level of ~ 100 nM compared to 1,2 mM in the
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extracellular concentration. To allow this, cells need different effectors like pumps, ion channels or exchangers to maintain the gradient [25].
Such complex patterns are generated by a coordinated interaction of calcium entry, calcium mobilization, calcium extrusion and calcium uptake mechanisms under the control of extracellular agonists and intracellular receptor-coupled second messenger systems. On the other hand, cell death is usually associated with global, sustained calcium increase due to excessive calcium entry and mobilization commonly accompanied by the prolonged decrease in the ER calcium content [1] [26]. Because cancer is associated with the altered endogenous expression of Ca2+-handling proteins and/or Ca2+-regulated effectors, this, results in the disruption of normal calcium signalling and deregulation of life as well as death-related processes ultimately leading to the development of certain malignant phenotypes.
1.Plasma membrane calcium ATPases (PMCAs)
These actively extrude calcium from the cell and are essential components in maintaining intracellular calcium homeostasis. There are four PMCA isoforms (PMCA1-4), and alternative splicing of the PMCA genes creates over 30 distinct isoforms, which are expressed differentially in various cell types in normal and diseased states [27]. Different studies show that they are affected in various cancer model. Changes in PMCA expression have been reported in several forms of cancer [28] [29]. In breast cancer cell lines a modest upregulation of PMCA1 mRNA compared to cell lines derived from noncancerous tissue is documented, with pronounced up- regulation of PMCA2 mRNA in some breast cancer cell lines, such as ZR-75-1 and T47D cells [29] [30]. Breast cancer cell lines also tend to have lower levels of PMCA4 mRNA [31]. A recent study demonstrated that PMCA1knockdown augmented necrosis mediated by the calcium ionophore ionomycin, whereas apoptosis mediated by the inhibition of Bcl-2 (Bcl-2 belonging to the anti-apoptotic protein family activated by the intrinsic pathway ) was enhanced by PMCA4 silencing. PMCA4 silencing was also associated with an inhibition of NFκB nuclear translocation, and an NFκB inhibitor phenocopied the effects of PMCA4 silencing in promoting cell death induced by the inhibition of Bcl-2 [32]. Reduced expression of another PMCA isoform, PMCA2, augments ionomycin-mediated cell death in SH-SY5Y neuroblastoma cells [33], whereas its overexpression in T47D breast cancer cells bestows resistance on ionomycin- mediated cell death through the attenuation of [Ca2+]i responses [34] . The potential significance of this survival advantage is reflected in the poorer prognosis of breast cancer patients with elevated PMCA2expression [34]. Baggot et al have identified an inhibitory interaction between PMCA2 and the calcium-activated signaling molecule calcineurin in breast cancer cells which confer cell resistance to apoptosis [35]. Differentiation of HT-29 colon cancer cells is associated with an upregulation of PMCA4, consistent with augmented Ca2+ extrusion [36], whereas in human colon cancer samples early in the progression with cells becoming less differentiated, PMCA4 transcription decreases significantly. It was concluded that variations in PMCA4 expression and the associated remodeling of calcium efflux in colon cancer cells, provide a growth advantage, while avoiding apoptosis and conferring insensitivity to apoptotic stimuli [36].
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2. TRP Channels:
Members of the mammalian TRP family of ion channels are ubiquitously distributed and have an extraordinary diversity of gating mechanisms [37]. These enable them to mediate calcium entry in response to a variety of stimuli, including second messengers generated in response to surface receptor stimulation, mechanical stimuli coming from plasma membrane stretch as well as physical and chemical characteristics of the microenvironment.
TRPV6 is a highly calcium-selective channel protein expressed at the plasma membrane. Studies
have shown that this channel is strongly expressed in advanced prostate cancer and significantly correlates with the Gleason >7 grading, making it a strong marker of tumor progression and subsequent invasion of healthy tissues . Previous studies have shown that TRPV6 is involved in highly calcium-selective currents in prostate cells, and that it is tightly regulated by intracellular calcium concentrations [38] [39] [40]. During the progression of cancer the expression of TRPV6 is increased. This upregulation of TRPV6 has been demonstrated by Lehen’kyi et al. to be involved in the resistance to apoptosis observed in hormone-sensitive prostate cancer cells [41].
3. The ORAI family:
This family exists in three isoforms Orai1, 2 and 3 which have been shown to be involved in cancerogenesis. In physiological conditions Orai1 proteins are associated in homotetramere and form a calcium channel activated by the store depletion of the endoplasmic reticulum (ER) . They are activated in response to the surface receptor-stimulated mobilization of calcium from the ER stores and thereby provide calcium for refilling the ER store, as well as for signalling purposes. STIM1 is a single transmembrane domain protein, that is mostly localized in the ER membrane, serving as a [Ca2+]ER sensor through its luminal EF-hand Ca2+-binding domain. Following a decrease in [Ca2+]ER STIM1 redistributes into punctae close to the plasma membrane where it can interact with Orai1 plasma membrane Ca2+-permeable channel, thereby triggering its activation. In prostate cancer cells, previous studies have demonstrated the involvment of Store Operated Channel Entry (SOCE) in apoptosis induction [42]. During cancerogenesis in prostate cancer cells, Flourakis M et al demonstrated that expression of Ca2+-permeable Orai1 channel, expressed at the plasma membrane, is decreased. This modification of expression leads to the downregulation of SOCE (whose principal constituent is Orai1), which is responsible of the resistance to apoptosis [43].
In breast cancer cells, Orai3 can mediate calcium entry, contributing to intracellular calcium concentration. Faouzi M et al suggest that Orai3 participatees in apoptosis resistance [44]. These studies suggest that Orai protein’s expression level and association type could be important factors in the development of an apoptosis-resistant phenotype in cancerous cell models. A non- classical event involving Orai1 has also been described. Khadra N et al reported that clusterization of Orai protein could also lead to apoptosis resistance. Authors have shown that CD95, the adaptor protein Fas-associated death domain protein (FADD), colocalises with Orai1
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and triggers Orai1-mediated localized Ca2+ entry thereby preventing the activation of death signalling pathways [45].
B. In the cell
After crossing the plasma membrane, calcium is then free in the cytoplasm. Different possibilities exist : , calcium could either be re-uptaken by the endoplasmic reticulum or by the mitochondria, or could activate calcium-dependentprotein present in the cytoplasm.
1. Sarco/Endoplasmic Reticulum Ca2+ ATPase (SERCA):
The SERCA pump is the only calcium-uptake mechanism of the ER, which is why its function is a key to maintaining the ER luminal calcium content at an optimal level for both protein processing and Ca2+ signalling purposes. In humans, 3 genes (ATP2A1..3) generate multiple isoforms due to developmental or tissue-specific alternative splicing [46]. The most ubiquitous SERCA isoform is the ATP2A2 gene-encoded SERCA2, which is represented by three SERCA2a-c splice variants. While SERCA2a and SERCA2c are primarily expressed in the heart, SERCA2b is present in all tissues and is thought to be the major isoform of the ER Ca2+-pump. SERCA3, which has six splice isoforms, is often found to co-express with SERCA2b, but generally has more limited tissue distribution.
Downregulation of SERCA2b expression has also been shown to accompany the transition of prostate cancer (PCa) to the aggressive androgen-independent phenotype. This has been explained as one of the adaptive responses of androgen-independent PCa cells against ER stress- induced apoptosis via the maintenance of lowered ER Ca2+ filling [21] [22]. Thus, it seems that a reduced SERCA pump expression, irrespective of the mechanisms involved (epigenetic influences, mutations, altered activity of the transcription factors), is the common characteristic feature of all malignancies, whereby cancer cells resist apoptosis [47].
In Small Cell Lung Cancer (H1339) and Adeno Carcinoma Lung Cancer (HCC) cell lines, although not in Squamous Cell Lung Cancer (EPLC) andLarge Cell Lung Cancer (LCLC) cell lines, the ER Ca2+-content was lower than Normal Human Bronchial Epithelial (NHBE).
Bergner A et al have shown that a reduced Ca2+-content is correlated with reduced SERCA pump expression and a reduction in expression of calreticulin, which buffers calcium within the ER. Both reductions participate in increased apoptosis resistance [48]. In addition to these factors, , authors have highlighted in this Lung cancer model, the implication of an other molecular actor expressed on the ER, namely the IP3 Receptor.
2. The inositol 1,4,5-trisphosphate receptor (IP3R):
The primary calcium release channel on endoplasmic reticulum membranes is the inositol 1,4,5- trisphosphate receptor (IP3R). Deletion of the gene for IP3R results in apoptosis defects in response to multiple stimuli. Bergner A et al have demonstrated that IPR3 expression increases
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and participates in the fall in calcium levels in the ER, which is responsible for the apoptosis defect [48]. Conversely, augmented IP3R levels are associated with increased cell death [49]. A mechanistic basis for altered IP3R function during apoptosis was revealed with the discovery that cytochrome c binds to IP3R earlyin the apoptosis process. This interaction blocks the calcium- dependent inhibition of the IP3R function, resulting in increased calcium release from internal stores. An increase in calcium concentration in the cytoplasm together with mitochondrial calcium overload lead to the activation of apoptosis.
3. Transient receptor potential polycystic 2 (TRPP2):
The ion channel is mutated in autosomal dominant polycystic kidney disease (ADPKD) and protects cells from apoptosis by lowering the calcium concentration in the endoplasmic reticulum. ER-resident TRPP2 counteracts the activity of the sarcoendoplasmic Ca2+ ATPase by increasing the ER Ca2+ permeability. This results in diminished cytosolic and mitochondrial calcium signals upon stimulation of inositol 1,4,5-trisphosphate receptors as well as reducing calcium release from the ER in response to apoptotic stimuli. For the moment, an understanding is still lacking of the role of TRPP2 in the regulation of ER homeostasis and its implication in the induction of apoptosis or in the acquisition of a resistant apoptosis phenotype in cancer cell [50]. These actors are involved in the phenotype remodeling of cancer cells, and are responsible for the acquirement of an apoptotic resistant phenotype. Based on this evidence, modification to expression, activity or localisation could be used as a potential way to restore apoptosis. Yet these are not the only routes. Indeed some molecular actors which are not deregulated during cancer progression should be examined for this purpose, because of their specific activity in calcium- dependent apoptotic pathways.
A well-illustrated example is the plasmalemmal Na+/Ca2+ exchanger (NCX) that normally extrudes Ca2+ from the cell (forward mode), but is also able to bring Ca2+ into the cell (reverse mode) under special conditions such as intracellular Na+ accumulation or membrane depolarization. It has been shown that the reverse exchanger mode can be responsible for the increases in [Ca2+]i in several cancer cell types in response to the inhibitors of Na+/K+-ATPase pump, cardiac glycosides [51] [52], in HCT116 human colon cancer cells in response to the inhibitor of protein kinase C and sphingosine kinase , N,N-dimethyl-D-erythro-sphingosine [53].
II. Calcium transporting as potential target of chemotherapy
Despite compelling evidence concerning the disruption of calcium homeostasis in cancer cells leading to the promotion of some malignant phenotypes, as well as the identification of key Ca2+-transporting molecules with altered expression and/or function, the number of therapeutic approaches using calcium-transporting proteins as potential targets for chemotherapy are still limited, although some are promising.
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1. Calcium Channels and exchangers expressed on the plasma membrane as potential targets of chemotherapy:
The entry of calcium into the cell is an important component in apoptosis induction. Targeting channels or exchangers involved in this calcium entry is a way to potentiate apoptosis in the cell.
A. Calcium Channels :
Various calcium channels (i.e. TRP, SOC) are expressed on the plasma membrane and allow the influx of calcium into the cell. Store-operated calcium entry ( SOCE) allows the main entry of calcium in cells. Recent studie show that in breast cancer cells, interaction between CD95 (FasL) and Orai1 leads to resistance to apoptosis [45]. To overcome this deregulation, some teams suggest the development of recombinant CD95L molecules or agonistic monoclonal antibodies to TRAIL-receptor for therapeutic exploitation and some compounds are being tested in clinical trials in order to avoid such interractions as CD95/Orai1 [54] [55].
Nonetheless, CD95L is tightly-regulated and future therapeutical approaches may stem from studies addressing its post-translational modifications, such as processing by ADAM10 and SPPL2a (proteases that generate soluble FasL fragments), [56] [57] in order to prevent the resistance to apoptosis induced by Orai1 calcium entry.
TRPV6 is a member of the TRP channel family, necessary to maintain calcium homeostasis in the cell. In gastric cancer cells it has been demonstrated that TRPV6 could be activated by Capsaicin ( an organic compound extracted from chili peppers) and induces more apoptosis than in normal gastric cells, by increasing mitochondrial permeability by the activation of Bax and p53 in a JNK-dependent manner [58].
B. Plasma membrane exchangers:
Recently, studies have shown the interesting clinical use in cancer therapies of increasing contractile force in patients with cardiac disorders using Cardiac Glycosides such as Oleandrin, Ouabain, and Digoxin. Cardiac Glycosides are involved in the inhibition of the plasma membrane Na+/K+-ATPase, leading to alterations in intracellular K+ and Ca2+ levels. These glycosides are also able to stimulate and sustain calcium increase and apoptosis in androgen-independent metastatic human prostate adenocarcinoma cells. Cell death is associated with the early release of cytochrome c from mitochondria, followed by proteolytic processing of caspases 8 and 3 [59]. Xu ZW et al show that Ouabain ( inhibitor of Na+/K+-ATPase α1 ) in a hepatocellular carcinoma (model HCC) could induce apoptosis of HepG2 cells by an intracellular calcium increase associated with an increase in ROS production by the mitochondria [60].
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Others channels could be used as therapeutic targets, such as the voltage-gated potassium channel. Wang W et al demonstrate that 4-aminopyridine (4-AP) induces apoptosis of human acute myeloid leukemia ( AML) cells by raising [Ca2+]i through the P2X7 receptor pathway. 4- AP, a voltage-gated potassium channel blocker, has been identified as exerting critical pro- apoptotic properties in various types of cancer cells, by inducing a significant increase in [Ca2+]i, through the P2X7 receptor. This calcium overload leads to a disruption of the mitochondrial membrane potential and an activation of caspase 3 and 9 [61].
A recent study, using xenografts of colorectal cancer treated with Sulindac and Celecoxib ( non- steroidal anti-inflammatory drugs (NSAIDs)), shows the ability of these compounds to increase [Ca2+]i one of the consequences of which, is the activation of the intrinsic apoptosis pathway through the activation of calpain 9 [62].