Calcium in tumour metastasis: new roles for known actors

Tissue invasion and metastasis represents one of the six initial cancer hallmarks, as proposed by Hanahan and Weinberg1. Metastasis is generally defined as the spread of malignant cells from the primary tumour through the circulation to establish secondary growth in a distant organ. The ability to migrate is a prerequisite for a can-cer cell to escape the primary tumour and to enter the circulation2. In fact, mortality in cancer is mainly caused by metastatic dissemination from the original tumour to multiple tissues, and these metastases are often impos-sible to eradicate. Although directional movement is a property of all cells during development, tissue remod-elling and regeneration, the transition from a benign to a malignant phenotype reflects the invasive capacity that is gained by tumour cells and is a key hallmark of aggressive tumours.

The ubiquitous second messenger calcium is one of the crucial regulators of cell migration3. Although increases of intracellular Ca2+ concentration ([Ca2+]i) organized in space, time and amplitude have long been known to be involved in cell migration, the sources of Ca2+ and the mechanism by which it modulates this process in cancer cells are only now beginning to be understood. In this Review, we focus on how our understanding of the Ca2+ dependence of pro-metastatic behaviours has advanced with recent findings that have outlined the function of the Ca2+-permeable transient receptor potential (TRP) channels, as well as the newly identified constituents of store-operated calcium (SOC) channels, the calcium release-activated calcium channel protein 1 (ORAI1) and stromal interaction molecule 1 (STIM1), in metastasis.

Sources of cytosolic calciumIncreases of [Ca2+]i can occur as a result of Ca2+ entry from the extracellular space and Ca2+ liberation from intracellular sources, mainly from the endoplasmic reticulum (ER). Both Ca2+ entry and Ca2+ release are pre-cisely controlled by numerous regulatory systems that provide the specific spatial and temporal characteristics of an intracellular calcium signal that are required for sustaining certain cellular functions4.

Ca2+ entry pathways. There are five major classes of plasma membrane Ca2+-permeable channels, which are differentially expressed in various cell types and which mediate Ca2+ entry in response to various activating stimuli. The first to be described were voltage-gated calcium channels (VGCCs), which belong to the Cav family5, and which are activated by depolarizing mem-brane potentials and are mostly expressed in excitable cells. Ligand-gated channels, which include representa-tives from the Cys-loop, glutamate and P2X purinergic ionotropic receptor families6, are mainly involved in fast chemical synaptic transmission in the nervous system, but can also be found extrasynaptically, as well as out-side the nervous system (particularly, P2X channels). Members of the mammalian TRP family of ion channels stem from a Drosophila melanogaster orthologue that is involved in visual transduction7. TRP channels are ubiq-uitously distributed and have an extraordinary diversity of gating mechanisms8, which enables them to mediate Ca2+ entry in response to various stimuli, including second messengers that are generated in response to surface receptor stimulation; stretching of the plasma

*INSERM, U1003, Laboratoire de Physiologie Cellulaire, Equipe labellisée par la Ligue contre le cancer, Villeneuve d’Ascq, F‑59650, France, and Universite de Lille 1, Villeneuve d’Ascq, F‑59650 France.‡Bogomoletz Institute of Physiology and International Center of Molecular Physiology, NASU, Bogomoletz Str., 4, 01024 Kyiv‑24, Ukraine.Correspondence to N.P. e‑mail: Natacha.Prevarskaya@univ‑lille1.frdoi:10.1038/nrc3105

Depolarizing membrane potentialsAll types of resting cells are characterized by the negative potential on their plasma membrane, thus the interior of the cell is negatively charged with respect to the extracellular space. The shift of the membrane potential to less negative values relative to the resting potential is called depolarization and the shift to more negative values is called hyperpolarization.

Calcium in tumour metastasis: new roles for known actorsNatalia Prevarskaya*, Roman Skryma* and Yaroslav Shuba‡

Abstract | In most cases, metastasis, not the primary tumour per se, is the main cause of mortality in cancer patients. In order to effectively escape the tumour, enter the circulation and establish secondary growth in distant organs cancer cells must develop an enhanced propensity to migrate. The ubiquitous second messenger Ca2+ is a crucial regulator of cell migration. Recently, a number of known molecular players in cellular Ca2+ homeostasis, including calcium release-activated calcium channel protein 1 (ORAI1), stromal interaction molecule 1 (STIM1) and transient receptor potential (TRP) channels, have been implicated in tumour cell migration and the metastatic cell phenotype. We discuss how these developments have increased our understanding of the Ca2+ dependence of pro-metastatic behaviours.

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© 2011 Macmillan Publishers Limited. All rights reserved

mailto:[email protected]

mailto:[email protected]

GatingThe process of opening and closing ion channels by various stimuli.

membrane; and physical and chemical characteris-tics of the microenvironment. The SOCs that require STIM1 and ORAI1 for operation9,10 are the major Ca2+ entry pathways in non-excitable cells and are widely distributed in various cell types. They are activated in response to surface receptor-stimulated mobilization of Ca2+ from the ER stores and thus provide Ca2+ for refill-ing of the ER store, as well as for signalling purposes. STIM1 is a single-transmembrane domain protein that is mostly localized to the ER membrane. It has an EF-hand Ca2+-binding domain through which it senses the lumi-nal Ca2+ concentration. Following a decrease in the luminal Ca2+ concentration, STIM1 redistributes into punctae close to the plasma membrane where it can interact with ORAI1, which is a four-transmembrane domain plasma membrane Ca2+-permeable channel. This interac-tion activates ORAI1 resulting in an inward Ca2+ current into the cell. Although the STIM1 homologue, STIM2, bears a clear resemblance to STIM1 in overall structure and basic properties, such as ER localization, luminal Ca2+ binding and redistribution to puncta at ER–plasma membrane contacts upon Ca2+ store depletion, its role in store-operated Ca2+ influx remains controversial10.

Finally, arachidonate-regulated Ca2+ (ARC) entry channels, which are activated in response to receptor-mediated derivation of arachidonic acid11, are primarily involved in the generation and modulation of agonist-induced oscillatory [Ca2+]i signals in some cell types (such as, various cell lines, parotid cells and pancreatic acinar cells). According to recent data, the same proteins that form SOC channels are also integral components of ARC channels; however, there are mechanistic differences between the channels. First, activation of the ARC chan-nels depends on a pool of STIM1 that is constitutively

present in the plasma membrane; and second, the pore of the ARC channels is formed by the heteromeric assembly of ORAI1 with its homologue ORAI3. How arachidonic acid activates the channels remains unclear12.

This list of Ca2+-permeable channels is probably incomplete. Indeed, a novel type of constitutive, store-independent Ca2+ entry channel formed by ORAI1 and the secretory pathway Ca2+-ATPase 2 (SPCA2; also known as ATP2C2) was recently proposed13.

Ca2+ mobilization. Mobilization of Ca2+ from the ER stores is achieved through two principal mechanisms: calcium-induced calcium release (CICR) and surface G protein-coupled receptor (GPCR)-stimulated calcium liberation14. Genuine CICR involves the gating of the ER membrane Ca2+ release channels, known as ryanodine receptors (RYRs), by cytosolic Ca2+. However, RYRs can also be gated through protein–protein interactions with plasma membrane VGCCs (this gating is characteristic of skeletal muscle) and through the cytoplasmic messenger cyclic ADP-ribose (cADPR)15,16. Agonist-mediated stimulation of Gq-coupled GPCRs induces inositol phospholipid breakdown by phospholipase C (PLC), which generates two important second messengers, diacylglycerol (DAG) and inositol trisphosphate (IP3; also known as Ins(1,4,5)P3). DAG can regulate protein kinase C (PKC), as well as some TRP channels directly, and IP3 can bind to the ER membrane Ca2+-permeable IP3 receptor channels (IP3R) and cause them to open, releasing the stored Ca2+ into the cytosol17.

Recent studies have unveiled an additional Ca2+ liber ation mechanism from endosomes and lysosomes through a novel class of endolysosomally localized nicotinic acid adenine dinucleotide phosphate (NAADP)-sensitive two-pore channels (TPCs)18. Ca2+ that is locally released from endolysosomes can further be coupled to the Ca2+ release from the ER through CICRs, thereby inducing global calcium signals. Notably, in lymphokine-activated killer (LAK) cells NAADP was shown to increase lysosome-related [Ca2+]i signalling, as well as cell migration, suggesting a role of NAADP in Ca2+-dependent migratory behaviour19.

Calcium and metastatic behavioursTo become invasive, tumour cells need to acquire capacities that enable enhanced migration through, and proteolysis of, the extracellular matrix (ECM). Specialized membrane protrusions provide several functions: lamellipodia are cytoskeletal actin pro-jections on the mobile edge of the cell, and invado-podia are proteolytically active plasma membrane protrusions that are responsible for the focal degra-dation of ECM components. Invadopodia are most common in highly invasive cancer cells and they contain concentrated foci of ECM-degrading matrix metalloproteinases (MMPs)20,21.

Polarized, migrating cells exhibit a stable and tran-sient gradient of [Ca2+]i, increasing from the front of the cell to the rear, that is thought to be responsible for rear-end retraction22,23. Such retraction is sup-ported by myosin II contraction, which is regulated by

At a glance

•TheubiquitoussecondmessengerCa2+isacrucialregulatorofcellmigration.AlthoughincreasesinintracellularCa2+concentration([Ca2+]

i)organizedinspace,

timeandamplitudehavebeenknowntobeimportantincellmigrationforsometime,thesourcesofCa2+andthemechanismbywhichitmodulatesthisprocessincancercellsareonlynowbeginningtobeunderstood.

•Ingeneral,Ca2+-dependentmechanismsofmalignantmigrationdonotseemtobeverydifferentfromthosethatareevidentinnormalphysiologicalmigration,andthussearchingforpotentialdifferencesisquiteachallengingtask.ThemajordifferencesseemtoariseonaquantitativelevelowingtoaberrantexpressionofCa2+-handlingproteinsand/orCa2+-dependenteffectors,leadingtotheincreasedturnoveroffocaladhesionsandmoreeffectiveproteolysisofextracellularmatrixcomponents.

•Recently,anumberofknownmolecularplayersincellularCa2+homeostasis,suchastheCa2+-permeablemembersofthetransientreceptorpotential(TRP)channelfamilyandtheconstituentsofstore-operatedCa2+entry,calciumrelease-activatedcalciumchannelprotein1(ORAI1)andstromalinteractionmolecule1(STIM1),havebeenimplicatedinthedevelopmentofthemetastaticcellphenotypeandtumourcellmigration.ThedatalinkingspecificTRPchannelstocancercellmigration,invasionandmetastasisarestilllargelyphenomenological.

•Ca2+-permeableionchannelsmightalsobeofuseindeterminingprognosis,astheexpressionpatternofsuchchannels,andthedegreeoftheirfunctionality,changewithcancerprogression.

•Ca2+signallingstillconstitutesanovelareaofresearchinoncology.Asthisfieldisstillratheryoung,notallthepotentialplayershaveyetbeeninvestigated,andforthosethathavebeenstudied,thespecificrolesinmigration,invasionandmetastasisofdifferenttypesofcancersareonlyjustbeginningtobeunderstood.

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p130CAS

Nature Reviews | Cancer

ER store

RYR

IP3R

Ca2+ Ca2+-dependenteffectors

Actin stressfibres

Zyxin

α-actinin α-actinin

α-actinin α-actinin

Vinculin

VinculinVinculin

FAK FAK

SRC SRC

Integrins

p130CAS

Channel

ECM

Myosin Myosin Myosin

Paxillin

Talin

Paxillin

Talin

α β αβ

myosin light-chain (MLC) phosphorylation through Ca2+-dependent MLC kinase (MLCK) and by disas-sembly of adhesions at the rear of the cell owing to calpain-mediated cleavage of focal adhesion proteins such as integrins, talin, vinculin and focal adhesion kinase (FAK)24. However, the cyclic morphological and adherence changes that are observed during cell migra-tion are also accompanied by repetitive Ca2+ signals, which take the form of Ca2+ spikes or oscillations25. Both Ca2+ release and Ca2+ influx have been linked to cell migration depending on cell types and stimuli26,27.

Ca2+-dependent components of cell migration and invasion. The coordinated and dynamic formation and disassembly of cell adhesions with the ECM is required for cell migration. Central to this process are structural and signalling linkage points between the ECM and the cytoskeleton that are known as focal adhesions. The main components of focal adhesions are integrin adhesion receptors that span the cell membrane to link the ECM with numerous intracellular molecules, including the actin cytoskeleton (FIG.1). The speed of the

formation and the disassembly of focal adhesions, which is known as focal adhesion turnover, determines how efficiently a cell migrates.

A prominent component that is involved in the regulation of focal adhesion turnover is FAK28, which is a non-receptor tyrosine kinase that promotes cell migration by coordinating signals between integrins and growth factor receptors. FAK is regulated by phos-phorylation at multiple tyrosine and serine residues, which makes it a point of convergence and integration of numerous extracellular stimuli, including those medi-ated through increases in [Ca2+]i and the recruitment of Ca2+/calcineurin-dependent29 or Ca2+/calmodulin-dependent protein kinase II (CaMKII)-dependent30,31 pathways. The association of FAK with focal adhesion sites and the autophosphorylation of FAK correlates with discrete local increases in [Ca2+]i, suggesting that Ca2+ prolongs the residency of FAK at focal adhesions, pos-sibly through tyrosine phosphorylation, thereby increas-ing the activation of its effectors, which are involved in focal adhesion disassembly32. FAK is also one of the substrates of a Ca2+-dependent protease, calpain33, which probably contributes to the Ca2+-mediated regulation of normal and pathological adhesion dynamics.

The effect of Ca2+ on focal adhesion turnover could also be mediated through calcineurin, which is involved in the polarized Ca2+-dependent recycling of integrins from adhesion complexes that have been released at the rear of a migrating cell to the leading edge34. A potential biochemical link between [Ca2+]i and the focal adhesion complex is also provided by focal adhesion-localized proline-rich tyrosine kinase 2 (PYK2), which requires Ca2+ for activation35. The regulation of intestinal epithelial cell migra-tion by chemokine GPCRs was shown to involve calcium-mediated activation of PYK2 (REF. 27).

S100A4, a member of the S100 family of EF-hand calcium-binding proteins, is also important in Ca2+-dependent metastatic pathways36. S100A4 is localized to the nucleus, cytoplasm and the extracellular space, and it predominantly exists in cells as a symmetric homodimer that enables the simultaneous binding and the functional crosslinking of two target proteins in a calcium-dependent manner. Calcium-dependent regulation of S100A4 involves a conformational shift that occurs on calcium binding and exposes interaction domains that are required for its interaction with several protein partners. Although S100A4 is not directly impli-cated in the formation of focal adhesions, its interaction with cytoskeletal proteins, including actin, nonmuscle myosin IIA, nonmuscle myosin IIB and tropomyosin, results in increased cell migration37. S100A4 was shown to colocalize with its most common interaction partner, myosin heavy chain IIA, to lamellipodia structures in migrating breast cancer-derived cells38. Other molecular targets of S100A4 action involve the leukocyte com-mon antigen-related (LAR), transmembrane protein tyrosine phosphatase-interacting protein liprin β1 and the tumour suppressor protein p53 (REF. 37). S100A4 has also been implicated in epithelial–mesenchymal transition (EMT)39. Moreover, there is evidence that

Figure 1 | Molecular organization of focal adhesion complexes and their regulation by Ca2+. Integrins are α- and β-transmembrane heterodimeric proteins that function as adhesion receptors and that span the cell plasma membrane to link the extracellular matrix (ECM) with the cytoskeleton through numerous intracellular molecules81. The integrin-binding proteins paxillin and talin recruit focal adhesion kinase (FAK) and vinculin. Cytoskeletal protein α-actinin is phosphorylated by FAK, binds to vinculin, crosslinks actomyosin stress fibres and tethers them to focal contacts. Zyxin is an α-actinin and stress fibre-binding protein that is present in mature contacts. The membrane-associated protein tyrosine kinase SRC and the adaptor protein p130CAS associate with focal contacts following integrin clustering. Proteins that are transiently present at focal contacts, such as ERK2 and calpain, are not shown. Spatially confined sustained or transient increases of Ca2+ concentration can occur in the form of waves, sparks or flickers. Such increases can occur as a result of Ca2+ entry through plasma membrane Ca2+-permeable channels and Ca2+ liberation from an endoplasmic reticulum (ER) calcium store through ryanodine (RYR) and/or inositol trisphosphate (IP

3R) receptor

channels. These changes influence Ca2+-dependent effectors (kinases, proteases and phosphatases), which in turn regulate focal adhesion components, thus facilitating the formation or disassembly (that is, turnover) of focal adhesions.

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m-calpainIsoform of Ca2+-dependent cystein protease that is activated by ~millimolar [Ca2+]i. It differs from another isoform, μ-calpain, which is activated by micromolar [Ca2+]i.

S100A4 controls the invasive potential of human pros-tate cancer cells through the regulation of MMP9 expres-sion40, thus contributing to the invadopodia-mediated proteolytic degradation of ECM components. In HeLa and MDA-MB-231 cells, an increase in [Ca2+]i owing to mobilization from the ER stores, which is independent of the stimuli used, induces the translocation of S100A4 from mostly perinuclear regions to the cytoplasm and plasma membrane regions41, where it could interact with target proteins thereby promoting cell migration.

In general, Ca2+-dependent mechanisms of malignant migration do not seem to be very different from those that characterize normal physiological migration, and so searching for potential differences is quite a chall-enging task. The major differences seem to arise on a quantitative level owing to the aberrant expression of Ca2+-handling proteins and/or Ca2+-dependent effectors, leading to the increased turnover of focal adhesions and more effective proteolysis of ECM components. Specific Ca2+ signatures of metastasizing cells remain unclear, and as a result several important questions need to be addressed. Are global cytosolic Ca2+ increases manda-tory for cancer cell migration? What are the patterns of localized Ca2+ signalling events in the metastasizing cell? Are these patterns universal for all types of cancers or are they cancer cell-specific? Do these patterns arise from the activity of the same plasma and ER membrane Ca2+-permeable channels, irrespective of cancer type? Does the colocalization of Ca2+-entry channels and/or Ca2+-release channels with Ca2+-sensitive molecular compo-nents of cell migration change during the acquisition of metastatic potential? And, finally, does such colocaliza-tion determine the specific role of those channels in each step of the metastatic cascade? The currently available data provide only partial answers to these questions.

Ca2+ entry pathways in the modulation of cell migration and invasion. As cell migration is associated with local tensions in the cell membrane, stretch-activated Ca2+-permeable channels are thought to be important trans-ducers of local mechanical stress into an intracellular Ca2+ signal42,43. The membrane stretch-dependent mode of activation is characteristic of several channel types44, of which TRP family member TRPM7 was specifically implicated in Ca2+ signalling at the front of migrating cells45,46 (TABLE 1). Ca2+ signals that are responsible for the local events at the front of migrating fibroblasts take the form of [Ca2+]i flickers that spread over a diameter of 5 μm and last 20–2000 milliseconds (ms). They occur because of the interaction of TRPM7-mediated Ca2+ influx with Ca2+ release through type 2 IP3R and they guide migration towards a chemoattractant45,46 (FIG. 2a). Although these results were obtained in normal fibro-blasts, they may also be applied to cancer cells, as local membrane tensions at the front of the cell are common to all migrating cells. However, the specific involvement of TRPM7 in the transduction of mechanical stretch at the leading edge of the plasma membrane into local increases of [Ca2+]i in metastasizing cancer cells needs further proof given that the activation of several TRP channels can be modulated by mechanical force8.

Aside from its involvement in stretch-dependent signalling, TRPM7 is activated by decreased intra-cellular Mg-ATP levels and also has a kinase domain8. Overexpressed TRPM7 tends to colocalize with m-calpain at peripheral vinculin-containing adhesion complexes in HEK-293 cells, which enables the control of m-calpain activity through local Ca2+ influx and the regulation of cell adhesion through m-calpain-mediated disas-sembly or turnover of peripheral adhesion complexes47 (FIG. 2b). High TRPM7 expression levels promoted loss of cell adhesion, whereas TRPM7 silencing strengthened cell adhesion and increased the number of peripheral adhesion complexes in the cells47. On the basis of the results obtained in model systems one can speculate that the depletion of intracellular Mg-ATP under the pro-metastatic anoxic or hypoxic conditions that are typically found in tumours48 and the concomitant activation of TRPM7 might contribute to the enhanced motility of can-cer cells owing to the promotion of m-calpain-mediated turnover of peripheral adhesions (FIG. 2b).

A pro-migratory role for TRPM7 was also demon-strated in nasopharyngeal carcinoma (NPC) cells, in which impaired TRPM7 channel function significantly decreased cellular migratory potential; conversely, increased TRPM7 activity promoted migration49. Moreover, in this study, the activation of TRPM7 pro-duced a global increase in [Ca2+]i owing to both Ca2+ entry and CICR involving RYRs (FIG. 2b).

Other TRP members are also involved in metastatic migratory behaviour (TABLE 1). Increased migration is correlated with higher expression of TRPV1 in human hepatoblastoma (HepG2) cells50, TRPV2 in human pros-tate cancer cells51,52 and TRPM8 in human glioblastoma (DBTRG) cells53,54 (FIG. 2c). However, increased expres-sion of TRPM1 in melanoma cells55, which was impli-cated in the regulation of Ca2+ homeostasis in human melanocytes56, was associated with reduced metastatic and migratory potential.

The data linking specific TRP channels to cancer cell migration, invasion and metastasis are still largely pheno menological, and the Ca2+-dependent molecular components that are activated downstream of these chan-nels are often unknown. It has been shown that TRPM8 overexpression reduces the motility of PC-3 prostate cancer cells through the inactivation of FAK57; whereas, enhanced translocation of TRPV2 to the plasma mem-brane (induced by lysophospholipids (LPLs) through activation of Gi or Go proteins and phosphatidylinositol 3,4-kinase (PI3,4K) signalling51) and the resulting Ca2+ entry promotes PC-3 cell migration by induction of key invasion markers, such as MMP2, MMP9 and cathepsin B52 (FIG. 2a). Importantly, small interfering RNA (siRNA)-mediated TRPV2 silencing was also able to reduce the size and invasive properties of xenografted PC-3 prostate tumours in nude mice, as well as downregulate the expres-sion of MMP2, MMP9 and cathepsin B52, suggesting that TRPV2 is a viable anti-metastatic target in vivo.

The major Ca2+ entry pathway in non-excitable cells in general, and cancer cells in particular, is provided by SOC channels, which require STIM1 and ORAI1 to function9. These ubiquitous SOC constituents have recently been

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Table 1 | Plasmalemmal and endolemmal Ca2+-permeable channels in migration and metastasis

Channel family

Family member*

Activation or modulation Cell model Metastatic process Main effectors Refs

Calcium influx

TRP TRPC?‡ Ca2+ store depletion, mechanical stretch and PLC signalling pathway

HT1080 fibrosarcoma cells Increased motility and invasion

ND 64

TRPM1 Constitutively active B16-F1 melanoma cells Reduced metastasation ND 55

TRPM7 Membrane stretch activated and Mg-ATP inhibited

Fibroblasts Migratory cell leading edge guiding

ND 45, 46


Transfected HEK-293 cells Increased turnover of peripheral adhesions

m-calpain 47


NPC Increased migration ND 49

TRPM8 Cooling (<28 ºC), cooling agents (menthol and icilin), PIP

2 and LPLs

DBTRG glioblastoma cells Increased migration ND 53, 54

TRPM8 Cooling (<28 ºC), cooling agents (menthol and icilin), PIP

2 and LPLs

Transfected PC-3 prostate cancer cells

Decreased migration FAK inactivation 57

TRPV1 Heat (>43 ºC), vanilloids (capsaicin), anandamide and polyamines

HGF-treated HepG2 hepatoblastoma cells

Increased migration ND 50

TRPV2 Heat (>52 ºC), osmotic cell swelling and LPLs

PC-3 and transfected LNCaP prostate cancer cells and PC-3 xenograft tumours in mice

Increased migration and invasion

MMP2, MMP9 and cathepsin B

51, 52

SOC STIM1– ORAI1

Receptor-activated Ca2+ store depletion

MDA-MB-231 human breast cancer cells, 4T1 mouse mammary tumour cells and MDA-MB-231 xenograft tumours in mice

Increased migration and higher rate of focal adhesion turnover

Ras and Rac small GTPases

58

Non-SOC SPCA2– ORAI1

Constitutively activated Breast cancer cells Promotion of tumorigenesis

ND 13

Cav

L-type (Ca

v1?)

Plasma membrane depolarization

MEF, MDA-MB-231 human breast cancer cells and 4T1 mouse mammary tumour cells

Increased PDGF-induced migration owing to trailing end contraction

CaMKII- and MLCK-dependent phosphorylation of MLC

26

T-type (Ca

v3?)

Plasma membrane depolarization and window current§ at resting potential

HT1080 fibrosarcoma cells Increased motility and invasion

ND 64

Calcium release

IP3R IP

3R? GPCR-stimulated PLC and

IP3-mediated release

Swiss 3T3 fibroblasts Increased migration CaMKII-dependent FAK phosphorylation

30

IP3R? EPAC-activated PLC and

IP3-mediated release

Melanoma cells Increased migration Actin assembly 67

IP3R2 Chemoattractant- and

TRPM7-coupled PLC and IP

3-mediated release

WI-38 fibroblasts Migratory cell leading edge guiding

ND 45, 46

IP3R3 PLC- and IP

3-mediated

releaseGlioblastoma cells and mouse xenograft model of glioblastoma

Increased migration ND 65

RYR RYR? TRPM7-induced CICR NPC Increased migration ND 49

RYR? cADPR-induced release Human cervical (HeLa) and breast (MDA-MB231) cancer cells

Increased metastasation Induction of S100A4 41

cADPR, cyclic ADP-ribose; CaMKII, Ca2+/calmodulin-dependent protein kinase II; CICR, calcium-induced calcium release; EPAC, exchange proteins directly activated by cAMP; FAK, focal adhesion kinase; GPCR, G protein-coupled receptor; HGF, hepatocyte growth factor; IP

3, inositol trisphosphate; IP

3R, IP

3 receptor;

LPLs, lysophospholipids; MEF, mouse embryonic fibroblast; MLC, myosine light chain; MLCK, MLC kinase; MMP, matrix metalloproteinase; ND, not determined; NPC, nasopharyngeal carcinoma; ORAI1, calcium release-activated calcium channel protein 1; PDGF, platelet-derived growth factor; PIP

2, phosphatidylinositol

4,5-bisphosphate; PLC, phospholipase C; RYR, ryanodine receptor; SOC, store-operated calcium; TRP, transient receptor potential . *Designation of the members of protein (channel) families is provided in accordance with the International Union of Pharmacology (IUPHAR) Nomenclature Committee (see the IUPHAR website; see Further information). ‡When designation includes a question mark this means that the exact isoform of the protein has not been determined. §Current at membrane potentials at which a voltage-gated channel reaches some level of activation, but its inactivation is not complete.

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ER storeER store

Nature Reviews | Cancer

ER store

ECM

Metastatic cell

Leading edge

Focal adhesionsPeripheral adhesions

Trailing end

Cold, menthol, PIP2 and LPLs

Heat, capsaicine, anandamide and spermine

TRPM8

TRPM7

TRPV1 TRPC

CTSB

Heat

MMPsMembrane stretch

Membrane stretch

Ca2+-dependentphosphorylation ofcontractile proteins

VGCC

Membrane potential

Trailing end contraction

PLC-dependentsignalling

Enhanced migration

ER store

ab

cd

Leading edge guidance

TRPM7

Chemoattractant

IP3R

IP3R

Induction of MMPs and CTSB

LPLsTRPV2

Mg-ATPdepletion

CICR

m-calpain

FAK cleavage

Enhanced turnover ofperipheral adhesions

GPCR

DAG

Agonists

IP3

CaMKII

Enhanced actin assembly

FAK

Facilitated formation of focal adhesions

• CICR• cADPR

Stimulation ofRAS and RAC

Acceleratedturnover offocal adhesions

Cytoskeletalproteins

Enhancedmigration

Gq PLC

Epac

IP3R

IP3

S100A4

ORAI1

STIM1

P

Ca2+

Ca2+

Ca2+

Ca2+

Ca2+ Ca2+

Ca2+

Ca2+

Ca2+

Golgi

SPCA2

RYRRYR

Depletion

unexpectedly implicated in breast tumour cell migration in vitro and in a mouse model of metastases generated by tumour xenografts58 (TABLE 1). The inhibition of store-operated Ca2+ entry (SOCE) by a pharmacological agent, SKF96365, or by siRNA-mediated STIM1 or ORAI1

silencing was able to suppress MDA-MB-231 human breast cancer cell and 4T1 mouse mammary tumour cell migration, as well as reduce the spread of xenografted tumour cells in mice; whereas, heterologous over-expression of STIM1 and ORAI1 increased tumour cell

Figure 2 | Major Ca2+ entry and Ca2+ release systems involved in migration, invasion and metastasis. Different panels represent a magnified view of the events taking place within the areas of the migrating metastatic cell (shown in the middle of the figure). Lightning bolts indicate activating stimuli. At the leading edge (part a) membrane stretch-activated, transient receptor potential cation channel subfamily M member 7 (TRPM7)-mediated Ca2+ influx coupled to inositol trisphosphate receptor (IP

3R)-mediated Ca2+ release participates in the guidance of the leading edge towards a

chemoattractant45,46. Ca2+ influx through TRPV2 promotes migration by induction of key invasion markers, matrix metalloproteinase (MMP2), MMP9 and cathepsin B (CTSB)52. Activation of TRPM7 and associated Ca2+ influx at peripheral adhesions (part b) promotes m-calpain-mediated disassembly or turnover of peripheral adhesion complexes47, thus contributing to enhanced motility. At the trailing end (part c) Ca2+ influx through L-type voltage-gated calcium channels (VGCCs) regulates contraction through Ca2+-dependent phosphorylation of contractile proteins26, whereas certain types of Ca

v3 VGCCs64 and TRP members, TRPC1 (REF. 64), TRPM8 (REFS 53,54) and TRPV1 (REF. 50), are implicated in the enhanced

migration of cancer cells through unknown effectors. Formation of focal adhesions (part d) is facilitated by IP3R-mediated

Ca2+ release that is stimulated through surface G protein-coupled receptors (GPCRs) or exchange proteins directly activated by cAMP (EPAC) leading to Ca2+/calmodulin-dependent protein kinase II (CaMKII)-dependent focal adhesion kinase (FAK) phosphorylation30 or enhanced actin assembly67, respectively, by stromal interaction molecule 1 (STIM1) – calcium release-activated calcium channel protein 1 (ORAI1)-based store-operated Ca2+ entry, resulting in RAS and RAC activation58 and by secretory pathway Ca2+-ATPase (SPCA2)–ORAI1 complex-mediated constitutive Ca2+ influx13. IP

3R- and

ryanodine receptor (RYR)-mediated Ca2+ mobilization from the endoplasmic reticulum (ER) stores also promotes cell migration in an S100A4-dependent manner41. cADPR, cyclic ADP-ribose; CICR, Ca2+-induced Ca2+ release; DAG, diacylglycerol; ECM, extracellular matrix; LPLs, lysophospholipids; PLC, phospholipase C.

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invasion through Matrigel. The presence of STIM1- and ORAI1-mediated SOCE induced a higher rate of focal adhesion turnover and increased migration of metastatic cancer cells (FIG. 2d), whereas its reduction resulted in a larger size of focal adhesions, slowing down their turno-ver and consequently increasing adherence. The defects of focal adhesion turnover and cell migration induced by SOCE impairment could be rescued by constitu-tively active forms of the small GTPases RAS and RAC, suggesting the involvement of these regulators of focal adhesions in the modulation of cell migration by SOCE58. Interestingly, a recent paper showed that, in human breast cancers with the poorest prognosis, ORAI1-mediated cal-cium influx is remodelled owing to the altered expression of STIM1 and STIM2 (REF. 59). These data indicate that the STIM1/STIM2 ratio could be clinically relevant as a prognostic factor for human breast cancer.

STIM1–ORAI1-mediated SOCE was also shown to be crucial in platelet-derived growth factor (PDGF)-induced migration of rat aortic vascular smooth muscle cells (VSMCs)60. Moreover, STIM1–ORAI1 was found to be upregulated in VSMCs from injured vessels, which undergo substantial cellular remodelling, compared with non-injured vessels, suggesting the general importance of these SOC components in providing the Ca2+ that is required for increased cellular motility.

An entirely different signalling mechanism, in which Ca2+ influx through ORAI1 is activated independently of STIM1, has recently been described13. This mechanism involves the interaction of ORAI1 with SPCA2, and has been implicated in human breast cancer tumorigenesis13

(TABLE 1). SPCA2 is normally involved in Golgi Ca2+ sequestration; however, in breast cancer-derived cells and human breast tumours, its expression is increased (FIG. 2d). The suppression of SPCA2 attenuated basal Ca2+ levels and tumorigenic potential. The authors of this paper conclude that a SPCA2–ORAI1 complex elicits a novel type of constitutive store-independent Ca2+ signalling that promotes tumorigenesis13.

Interestingly, increased expression levels of STIM1–ORAI1-based SOCE seem to promote metastasis owing to the stimulation of breast tumour cell migration58. However, reduced expression of ORAI1-mediated SOCE was shown to contribute to the increased apop-tosis resistance in prostate cancer cells61. Thus, two of the cancer hallmarks1 — tissue invasion and metastasis and evasion of programmed cell death (apoptosis) — seem to require the opposite expression of STIM1–ORAI1-based SOCE. This conflicting result may indicate either dif-ferent Ca2+ dependence of cancer hallmarks in various types of cancers or the differential importance of global STIM1–ORAI1-mediated increases of [Ca2+]i (associated with apoptosis) rather than increases that are spatially restricted to focal adhesions (associated with migra-tion). It also calls for caution when considering STIM1–ORAI1-based SOCE as a potential therapeutic target in cancer treatment.

Although voltage-gated ion channels are not typi-cally present in cancer cells, increased metastatic potential is known to correlate with the expression of membrane channels and currents that are characteristic

of excitable membranes62, and sometimes even the background expression of the subtypes of VGCCs in apparently non-excitable cells can be sufficient to alter their motility (TABLE 1). Indeed, PDGF-induced migra-tion of fibroblasts was shown to require Ca2+ influx through L-type VGCCs26. The evidence suggested that this influx was necessary to regulate the contraction of the trailing end of migrating fibroblasts through CaMKII and MLCK-dependent phosphorylation of MLC26 (FIG. 2c). Despite angiotensin 1 receptor-coupled [Ca2+]i, signalling in human umbilical vein endothelial cells (HUVECs) was shown to involve the activation of both T-type (also known as Cav3) and L-type (also known as Cav1) VGCC; the stimulation of AT1 recep-tors promoted migration, which was sensitive only to the T-type channel blocker mibefradil63. The involve-ment of certain types of T-type channels (probably Cav3.1) was also suggested in the metastatic behaviour of HT1080 fibrosarcoma cells64. However, the motility and invasion of HT1080 cells, and accompanying dynamic Ca2+-signalling events, could be affected not only by mibefradil, but also by the blockers of non-voltage-operated Ca2+ channels carboxyamidotriazole (CAI) and gadolinium64. Non-voltage-operated Ca2+ channels are usually represented by the members of TRPC subfamily of TRP channels; therefore, based on these results, one can suggest the involvement of both T-channels and some TRPC members in the modulation of motility and invasion of HT1080 cells (FIG. 2c).

Ca2+ mobilization in the modulation of cell migra-tion and invasion. Ca2+ mobilization from intracellular stores amplifies the Ca2+ entry signal and determines its characteristics in space and time. The activation of surface GPCRs by bombesin, bradykinin or vasopressin in Swiss 3T3 fibroblasts causes Ca2+ mobilization and induces rapid FAK phosphorylation at Ser-843 through a Ca2+–calmodulin–CaMKII-dependent pathway30 (FIG. 2d). This phosphorylation site, together with three others (at Ser-722, Ser-732 and Ser-910), is located on the carboxy-terminal region of FAK in close proximity to the protein–protein interaction domains, suggesting a role for serine phosphorylation, and consequently of Ca2+ signalling, in modulating the binding of downstream signalling proteins.

Agonist-induced stimulation of surface GPCRs in glioblastoma cells evokes IP3-dependent Ca2+ mobili-zation through type 3 IP3R (IP3R3), the expression of which is aberrantly increased compared with normal cells65 (TABLE 1). Small hairpin RNA (shRNA)-mediated silencing of IP3R3 suppressed the [Ca2+]i increases and migration of glioblastoma cells65. Paradoxically, similar effects could be achieved with caffeine, which is com-monly known to be an activator of RYRs, but which has also been shown to inhibit the opening of IP3Rs66. Treatment with caffeine greatly increased the mean survival time in a mouse xenograft model of glio-blastoma, prompting the authors to suggest that IP3R3 is a novel therapeutic target and that caffeine is a possible adjunct therapy for slowing the invasive growth of glioblastoma65.

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Auxiliary (regulatory) subunitsA protein complex of ion channels can sometimes include additional subunits which do not participate in pore formation, but which modulate functional properties of the channel.

The PLC–IP3R Ca2+ release pathway has also been implicated in metastatic melanoma. Metastatic mela-noma cells express higher levels of the novel cyclic AMP sensor exchange proteins directly activated by cAMP (EPAC) compared with primary melanoma cells. It has recently been shown that increased melanoma cell migration involves EPAC-activated Ca2+ release from the ER through the PLC–IP3R pathway, which in turn promotes increased actin assembly67 (FIG. 2d). Thus, EPAC, in addition to the components of the PLC–IP3R Ca2+ release pathway, also represent potential targets for the suppression of melanoma cell migration and, conse-quently, the development of metastasis. Interestingly, the contact of melanoma cells with HUVECs triggers a rapid PLC–IP3-mediated [Ca2+]i response, altering endo thelial adherens junctions and enabling melanoma cells to breach the endothelium and enter the vasculature68. The disruption of endothelial cell junctions following contact with melanoma cells is shown by changes in the immu-nological staining patterns of vascular endothelial (VE)–cadherin and melanoma cell-induced VE–cadherin reorganization.

In addition, an increase in [Ca2+]i owing to mobili-zation from the ER stores can promote the migration of HeLa and MDA-MB-231 cancer cells through the Ca2+-dependent activation of S100A4, which enables its interaction with target proteins41 (FIG. 2d).

Clinical implications and concluding remarksImpaired calcium signalling is an important factor in the metastatic behaviour of cancer cells. There are signs of promising developments in targeting the molecular constituents of calcium signalling for restraining meta-static spread. Carboxyamidotriazole (CAI) is a cytostatic inhibitor of non-voltage-operated calcium channels and calcium channel-mediated signalling pathways, which suppresses angiogenesis, tumour growth, invasion and metastasis, and which is already in clinical trials as an orally active antineoplastic agent (see ClinicalTrials.gov; see Further information)

As the molecular machinery for calcium signalling is ubiquitous, selective targeting of its components in metastasizing cancer cells remains the major challenge for therapeutic use, as pharmacological impairment of any of these components would probably produce sig-nificant toxicity to normal cells. A possible strategy for circumventing this problem may be the coupling of a drug to a targeting moiety in order to produce a drug derivative that can only be activated within tumours. A good example of such a strategy is the ‘smart bomb’ for prostate cancer, which combines a sarcolemmal and endoplasmic reticulum Ca2+-ATPase (SERCA) inhibi-tor thapsigargin (which induces apoptosis through the activation of ER stress and Ca2+ entry pathways) with a targeting peptide that is a substrate of the serine/protease prostate-specific antigen (PSA)69. This prodrug has been shown to be toxic only to PSA-producing prostate cancer cells.

Additional complications in targeting various molecular components of calcium signalling for cancer therapies arise from the fact that selective

pharmacological tools for many of them are sim-ply not available. Therefore, new strategies based on siRNA and antisense technologies should be considered. Many plasmallemal and endollemal Ca2+-permeable ion channels and transporters are characterized by the presence of alternatively spliced variants70,71, which may be differentially expressed in normal and cancer cells, thus providing the possibil-ity for selective targeting. For example, the expres-sion of a novel glioma-specific T-type Ca2+-channel splice variant, Cav3.1ac, has been described in human glioma72. This variant was detected in biopsy samples and some glioma cell lines, but not in normal brain or fetal astrocytes, suggesting that it might contribute to tumour pathogenesis. Human glioma is also char-acterized by the expression of the specific splice vari-ant of large conductance Ca2+-dependent K+ channel (BK channel), which has enhanced Ca2+ sensitiv-ity73. An embryonic and neonatal splice variant of a voltage-gated sodium channel, Nav1.5e, is implicated in the enhancement of the migration and invasion of breast cancer cells74. Special attention should be paid to identifying more cancer-specific channel splice var-iants and their auxiliary (regulatory) subunits to enable selective targeting.

Plasma membrane Ca2+-permeable channels also have considerable potential for antibody-based targeting, especially those that have limited background expres-sion but that are strongly overexpressed in tumours and metastases. Even if anti-channel antibodies do not pro-duce functional effects, they could be used as carriers for radionuclides, toxic molecules or nanoparticles. Moreover, the use of fluorescently labelled antibodies against pro-metastatic Ca2+-permeable channels also has the potential for the detection and visualization of tumours and metastases.

The importance of Ca2+-permeable ion channels is not limited to cancer therapies. As the expression pattern of such channels, and the degree of their functional-ity, change in cancer, they may be useful for diagnostic purposes. A good example of this is the highly Ca2+-selective TRPV6, the expression and function of which was shown to correlate with prostate cancer grade75,76. Although TRPV6 was not directly linked to the depend-ence of tumour cell migration on Ca2+, it may be con-sidered as a truly pro-metastatic channel, as patients with TRPV6-positive prostate cancer were shown to have a higher potential for metastasis and tissue inva-sion beyond the prostate. Expression levels of TRPM8 might also be useful for identifying the stage of prostate cancer77,78. Decreased expression of TRPM1 has been shown to correlate with melanoma cell transition from a low to a high metastatic phenotype55, whereas tran-scriptional profiling of primary breast cancer specimens using DNA microarrays has identified that alteration in the ratio of STIM1 to STIM2 is relevant in poor breast cancer prognosis59.

Ca2+ overload is lethal to all cells, thus using gene transfer technologies to heterologously overexpress Ca2+-permeable channels in cancer cells in order to promote lethal Ca2+ influx is also of potential

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therapeutic importance. The feasibility of such an approach has been shown by selectively expressing a mutated Na+-permeable MDEG (or ASIC2) channel in gastric cancer cells in a mouse peritoneal dissemi-nation model of metastasis79. The constitutive Na+ influx that is mediated by mutated MDEG impairs normal intracellular ionic homeostasis and causes cell death80. Increased efficiency in the killing of tumour cells would be expected from the overexpres-sion of constitutively open isoforms of Ca2+-selective channels.

Ca2+ signalling still constitutes a novel area of research in oncology. As this field is still rather young, not all the potential players have been investigated, and for most of the players that have been studied, the specific roles in migration, invasion and metastasis of different types of cancers are only just beginning to be understood. With these, and other developments in cancer biology, it is expected that a more detailed understanding of the roles of Ca2+ signalling in the key processes involved in can-cer spread will facilitate the development of improved molecularly targeted tools for diagnosis and treatment.

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AcknowledgementsThe research of N.P. and R.S. is supported by grants from Institut National de la Santé et de la Recherche Médicale (INSERM), Ligue Nationale Contre le Cancer, Fondation de Recherche Medicale (FRM), Association pour la Recherche sur le Cancer (ARC) and Région Nord/Pas-de-Calais. Y.S. was supported in part by a visiting scientist program of the Universite de Lille 1.

Competing interests statementThe authors declare no competing financial interests.

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