doi:10.1152/ajpcell.00280.2011 302:C9-C15, 2012. First published 10 August 2011;Am J Physiol Cell Physiol

Alessandra Fiorio Pla, Daniele Avanzato, Luca Munaron and Indu S. AmbudkartargetsVascularizing the tumor: TRP channels as molecular Ion channels and transporters in cancer. 6.

You might find this additional info useful...

71 articles, 32 of which can be accessed free at:This article cites http://ajpcell.physiology.org/content/302/1/C9.full.html#ref-list-1

including high resolution figures, can be found at:Updated information and services http://ajpcell.physiology.org/content/302/1/C9.full.html

can be found at:AJP - Cell Physiologyabout Additional material and information http://www.the-aps.org/publications/ajpcell

This infomation is current as of December 28, 2011. 

American Physiological Society. ISSN: 0363-6143, ESSN: 1522-1563. Visit our website at http://www.the-aps.org/.a year (monthly) by the American Physiological Society, 9650 Rockville Pike, Bethesda MD 20814-3991. Copyright © 2012 by the

is dedicated to innovative approaches to the study of cell and molecular physiology. It is published 12 timesAJP - Cell Physiology

on Decem

ber 28, 2011ajpcell.physiology.org

Dow

nloaded from

Ion channels and transporters in cancer. 6. Vascularizing the tumor: TRPchannels as molecular targets

Alessandra Fiorio Pla,1,2,3 Daniele Avanzato,1 Luca Munaron,1,2,3 and Indu S. Ambudkar4

1Department of Animal and Human Biology, 2Center for Complex Systems in Molecular Biology and Medicine (SysBioM),3Nanostructured Interfaces and Surfaces Centre of Excellence, University of Turin, Torino, Italy; and 4Secretory PhysiologySection, Molecular Physiology and Therapeutics Branch, National Institute of Dental and Craniofacial Reserach, NationalInstitutes of Health, Bethesda, Maryland

Submitted 4 August 2011; accepted in final form 9 August 2011

Fiorio Pla A, Avanzato D, Munaron L, Ambudkar IS.Ion channels and transporters in cancer. 6. Vascularizing the tu-mor: TRP channels as molecular targets. Am J Physiol Cell Physiol302: C9 –C15, 2012. First published August 10, 2011;doi:10.1152/ajpcell.00280.2011.—Tumor vascularization is a criti-cal process that determines tumor growth and metastasis. In the lastdecade new experimental evidence obtained from in vitro and in vivostudies have challenged the classical angiogenesis model forcing us toconsider new scenarios for tumor neovascularization. In particular, thegenetic stability of tumor-derived endothelial cells (TECs) has beenrecently questioned in several studies, which show that TECs, as wellas pericytes, differ significantly from their normal counterparts atgenetic and functional levels. In addition to such an epigenetic actionof tumor microenvironment on endothelial cells (ECs) commitment,the distinct characteristics of TECs could be due to differences in theirorigin compared with preexisting differentiated ECs. IntracellularCa2� signals are involved at different critical phases in the regulationof the complex process of angiogenesis and tumor progression. Thesesignals are generated by a wide variety of intrinsic and extrinsicfactors. Several key components of Ca2� signaling including Ca2�

channels in the plasma membrane, endoplasmic reticulum, calciumpumps, and mitochondria contribute to the generation, amplitude, andfrequency of these Ca2� change. In particular, several members of thetransient receptor potential (TRP) family of calcium-permeable chan-nels have profound effects on the function of ECs. Because of itsmultifaceted role in the control of cell function, proliferation, andmotility, TRP channels have been suggested as a potential moleculartarget for control of tumor neovascularization. Since plasma mem-brane Ca2� channels are easily and directly accessible via the blood-stream, they are potential targets for a number of pharmacological andantibody-targeted therapeutic strategies, with specificity being themain limitation. In this review we discuss recent advances in under-standing the role of Ca2� channels, with specific reference to TRPchannels, in tumor vascularization process.

tumor vascularization; endothelial cells; calcium ions; transient recep-tor potential channels

TUMOR VASCULARIZATION provides blood supply for tumorgrowth and metastasis. It is now widely accepted that, inaddition to endothelial cells (ECs), which are the first mechan-ical and functional interface between blood and tissues, othercellular components are important in vessel formation, includ-ing pericytes, inflammatory cells, and other resident or circu-lating precursors. They contribute directly or indirectly to theregulation of multiple steps during which “activated” ECs

proliferate, migrate, differentiate, and are stabilized into a newcirculatory network (12, 25).

Although neovascularization occurs in different physiologi-cal and pathological processes such as embryo development,wound healing, cancer, cardiac ischemia, and diabetes, themechanisms underlying its genesis and regulation can differ,particularly in tumor progression. The most striking evidencesupporting this comes from the vascular targeting treatment(VTT) designed to damage tumor vasculature and inhibit localangiogenesis. Despite promising results, emerging data indi-cate that responses to VTT are short lived and resistancedevelops in the majority of patients (17, 63). The partialineffectiveness of VTT may be due to several reasons. Indeedan increasing amount of novel experimental evidence obtainedfrom in vitro and in vivo challenge the classical angiogenesismodel forcing us to consider new scenarios to explain theprocess of tumor neovascularization (10, 20, 33, 62, 66).

ECs were previously considered to be genetically stablecompared with tumor cells and therefore an ideal therapeutictarget in the development of anti-angiogenic treatments. How-ever, their stability has been questioned by several studiesshowing that tumor-derived endothelial cells (TECs) as well aspericytes differ significantly from their normal counterparts atgenetic and functional levels (10, 57, 67). Even macroscopicmorphological analysis demonstrates that tumor blood vesselsare irregular and dilated. Morphologically distinct venules,arterioles, and capillaries are undistinguishable (16, 26). Fromthe functional point of view this organization leads to a leakyand hemorrhagic network (16). TECs have been isolated andcultured from human carcinomas on the basis of membranemarkers and exhibit altered genotype, phenotype, and function.They are often aneuploid, display chromosomal instability, andhave distinct gene expression profiles that distinguishes themfrom normal ECs. In addition, TECs avoid senescence in vitro,a process typical of normal ECs, and instead display enhancedproliferation, motility, and the inherent ability to organize intocapillary-like tube formation (8, 10, 28).

That TEC characteristics are a result of their distinct originis a subject of strong debate. It has been suggested that whileTECs might have been derived from normal endothelium,further phenotypic changes could be induced by the tumormicroenvironment; e.g., by transfer of genetic material viaexosomes and/or microvesicles that are released from thetumor cells (42). This hypothesis is in agreement with thegeneral accepted idea that endothelium is a highly versatilesystem able to adapt to local tissue exigencies. Accordingly, ithas been reported that normal ECs cultured in the presence of

Address for reprint requests and other correspondence: A. Fiorio Pla, Dept.Animal and Human Biology, Univ. of Torino, V. Accademia Albertina 13,10123 Turin, Italy (e-mail: alessandra.fiorio@unito.it).

Am J Physiol Cell Physiol 302: C9–C15, 2012.First published August 10, 2011; doi:10.1152/ajpcell.00280.2011. Themes

http://www.ajpcell.org C9

on Decem

ber 28, 2011ajpcell.physiology.org

Dow

nloaded from

soluble tumor cell-derived factors undergo changes in thepattern of expression of specific proteins (32).

The other school of thought proposes that the peculiarity ofTECs could be due to their alternative origin, independent oflocal preexisting differentiated ECs. They may be the resultof vasculogenic events via differentiation of bone marrow-derived circulating stem cells, tissue-resident normal stemcells, or tumor stem cells (10, 12, 19). It could involve a sortof “recapitulation” of embryonic development. As aforemen-tioned, true “angiogenesis” occurs only in later phases ofdevelopment, being preceded by “vasculogenesis,” because ofthe ability of multipotent cells to differentiate into ECs (9, 10,12). These “stem cells” include different populations such ashemangioblasts, precursors of endothelial as well as hemato-poietic cells, and mesangioblasts that differentiate into ECs andmesodermal derivatives (bone, muscle, and cartilage). Theprimitive vasculature created by vasculogenesis is then remod-eled by angiogenesis.

A large amount of data strongly suggests that bone marrow-derived hematopoietic stem cells (HSCs) and non-HSC (mes-enchimal progenitors), together with pericytes and inflamma-tory cells (including tumor-associated macrophages, TAMs),could contribute to tumor vascularization. In particular, greatinterest has been focused on circulating endothelial progenitorcells (EPCs), a subpopulation of bone marrow-derived mono-nuclear cells also found in peripheral blood. They promotevessel formation in adult, and it was recently reported that theyhave the ability to incorporate into tumoral tissues (10). How-ever, the precise role and relevance of the different EC pro-genitors in tumor vascularization in vivo is still controversial(10, 12, 19). They could directly take part in vessel formationor/and act indirectly in a paracrine manner by stimulating ECsor by remodeling the extracellular matrix. On the basis of theseconsiderations, TECs represent a suitable model to investigatethe role of proangiogenic factors and their related cell signalingcompared with the widely used ECs obtained from normaltissues.

Ca2� Channels: Molecular Identities and Functional Role inTumor Vascularization

The role of Ca2� signals and channels in the cardiovascularfunction and disease is well recognized and subject of anumber of recent reviews (50, 54, 58, 72). Here we will focusour attention primarily on the proangiogenic Ca2� channelsthat have been described thus far. Several studies have high-lighted the importance of agonist-stimulated Ca2� signals inangiogenesis. Tyrosine kinase receptor-binding growth factorssuch as vascular endothelial growth factor (VEGF) and basicfibroblast growth factor (bFGF) stimulate intracellular Ca2�

([Ca2�]i) increase in ECs and trigger complex intracellularcascades. Cai

2� signals can be generated due to the release ofCa2� from intracellular stores, depletion of which leads toactivation of plasma membrane Ca2� entry via the store-operated Ca2� entry mechanism SOCE. An alternative route isstore-independent, receptor-operated Ca2� entry (ROCE). Themolecular nature of all the channels involved in these pro-cesses, especially in the latter instance, is not very well estab-lished, and several of these channel properties as well as themechanism of their activation remains to be determined.

The discovery of transient receptor potential (TRP) super-family of channels provided putative candidates for nonvolt-age-gated Ca2� entry mechanisms, including SOCE, or thoseactivated by mechanical stretch as well as intracellular mes-sengers such as 1,4,5-trisphosphate (InsP3) or arachidonic acid(AA) (6, 54). Several TRP channels have been identified in avariety of ECs, e.g., TRPC1, TRPC4, TRPC6, TRPM7,TRPV1, and TRPV4 (18, 41). They appear to be heteroge-neously expressed in different EC types and contribute toagonist-induced or mechanical stretch-induced Ca2� entry todifferent extents in these cells (18, 47). VEGF-mediated Ca2�

entry has been described in detail in human microvascular ECs(HMVECs). Tyrosine phosphorylation of VEGF Receptor 2(VEGFR2) triggers recruitment and activation of phospho-lipase C� (PLC�), InsP3, and diacylglycerol (DAG) genera-tion, and activation of TRPC3/TRPC6 channels via store-independent mechanism, which is not yet understood (14, 60).TRPC6 has been shown to be a critical component of thechannels mediating Ca2� entry in response to VEGF and1-oleoyl-2-acetyl-sn-glycerol (OAG, a membrane-permeantDAG analogue) in HMVECs. Expression of a dominant-neg-ative (inactive) mutant of the channel reduced cell proliferationas well as EC migration and sprouting in matrigel (29). Usinga similar approach, Ge and coworkers (27) proposed a criticalrole for TRPC6 in promoting cell proliferation as well as“capillary-like” structure formations in vitro induced by VEGF,but not bFGF, stimulation of HUVECs (see also Fig. 1).

Besides the role in EC proliferation, migration, and sprout-ing, TRPC channels and in particular TRPC6 have been alsoimplicated in the VEGF-DAG-mediated Ca2� entry and con-sequent increase in vascular permeability (Fig. 1). The increasein vascular permeability and [Ca2�]i induced by VEGF areindependent of calcium store release, as they are not affectedby thapsigargin. They are mimicked by OAG and can bepotentiated by TRPC6 agonist flufenamic acid (60). On theother hand, Malik’s laboratory provided evidence for a role ofTRPC1 in the increase of endothelial permeability (46, 56)(Fig. 1). VEGF has been suggested to activate these channelsvia Ca2� store depletion mechanism in HUVECs. In HMVECsanti-TRPC1 antibody blocked VEGF-induced SOCE and cellpermeability, while VEGF-mediated Ca2� signals were signif-icantly increased when TRPC1 was overexpressed. Because ofits role in EC permeability and its ability to oligomerize withTRPC1, the authors speculated that also TRPC4 could even-tually participate with TRPC1 in this process (39). The appar-ent variability in the type of VEGF-activated Ca2� channelsinvolved in regulation of EC permeability could be partiallyexplained by tissue differences, especially between small cap-illaries and large vessels. For example, the spectrum of theTRPC channels expressed in HMVECs is substantially differ-ent from that in HUVECs. TRPC4 is expressed in HUVECsbut not in HMVECs (29). Furthermore, a proangiogenic rolefor TRPC1 has been described in vivo. Knockdown of ze-brafish TRPC1 by morfolinos caused severe angiogenic defectsin intersegmental vessel sprouting, presumably due to impairedfilopodia extension EC migration. TRPC1 appears to be down-stream to VEGF in controlling angiogenesis and necessary toextracellular-regulated kinase (ERK) phosphorylation (74).

Recently, interesting data showed that in addition to thereported role for TRPC1 and TRPC6 in SOCE, Orai1, andStim1 (composing the CRAC channels) are key players in

Themes

C10 Ca2� CHANNELS IN TUMOR VASCULARIZATION

AJP-Cell Physiol • doi:10.1152/ajpcell.00280.2011 • www.ajpcell.org

on Decem

ber 28, 2011ajpcell.physiology.org

Dow

nloaded from

VEGF-mediated SOCE (1, 43). Both transient and sustainedphase of VEGF-evoked Ca2� entry are significantly reduced bysuppression of Orai1 or Stim1 expression in HUVECs (Fig. 1).VEGF stimulation promotes Stim1 clustering, a critical eventin activation of channels mediating SOCE such as Orai1 andTRPC1 (11, 43). Finally, a significant role for Orai1 in theangiogenic process both in vitro and in vivo was also demon-strated (1, 43). It is important to note that these data do not ruleout the possible contribution of TRPC1 to SOCE in these cells,especially when the functional interactions between Orai1 andTRPC1, which have been recently described, are taken intoconsideration (15, 34). It was reported that activation ofTRPC1 by STIM1 that occurs in response to store depletion isdependent on Orai1. Ca2� entry via Orai1 triggers recruitmentof TRPC1 to the membrane where it is gated by STIM1. Therole of Orai1 and STIm1 will not be elaborated in this review.However, it should be noted that Orai1 and TRP channels canconcertedly contribute to various functions of ECs and TECs.The relative roles of these channels in will have to be assessedwithin the context of a particular cell type and possibly specificcellular stimuli. The origin and culture of ECs can inducesignificant changes in the phenotype of the cells and thus is itcritical to take this point into consideration.

Different studies have suggested that TRPV4 is likely to be akey player in tumor vascularization and in particular cell migra-tion (Fig. 1). It is widely known that TRPV4 is widely expressedin the vascular endothelium where it is proposed to acts as amechanosensor. TRPV4 in ECs is activated in responses tochanges in cell morphology that occur during cell swelling or inresponse to shear stress. Fluid shear stress regulates cell reorien-tation in a TRPV4-dependent manner, whereas in larger arteriesthe channel is a key player in shear stress-induced vasodilation(30, 69). Endogenous compounds also regulate TRPV4 function.

AA, a 20-carbon fatty acid released by proangiogenic factors aswell as its metabolites, which are important inflammatory mes-senger, mediate EC migration toward sites of inflammation (18).Both shear stress and agonist activation of TRPV4 leads toenhancement of EC proliferation and triggers collateral growthafter arterial occlusion (70). A recent study reported by us dem-onstrated that expression of endogenous TRPV4 is significantlyhigher in tumor-derived EC (human breast carcinoma, BTEC)compared with “normal” ECs (HMVEC) (Fig. 1). Furthermore,AA-activated TRPV4 is essential for BTEC migration. Loss ofTRPV4 expression results in decreased Cai

2� responses to theTRPV4-specific agonist 4�-phorbol 12,13-didecanoate (4�PDD)and complete inhibition of AA-induced migration of BTEC. Themechanism by which AA regulates TRPV4 was also revealed.AA induces actin remodeling in BTECs, which triggers recruit-ment of TRPV4 in the plasma membrane of BTEC (23). What isinteresting is that TRPV4 and Ca2� increases is seen at the leadingedge of the BTEC. This suggests that AA stimulates local Ca2�

signaling, which drives migration of BTEC. It is important tostress that while this study used TECs to investigate the effect ofproangiogenic factors, a large number of previously reportedstudies have been performed in vitro on different types of primaryor immortalized macrovascular and microvascular EC lines fromhuman or animal normal tissues. In fact, study of proangiogeniccalcium signaling in TECs has been initiated only very recently(2, 21, 22, 49, 51). Notably the functional relevance of AA-induced calcium entry could be restricted to tumor-derived ECs,since AA evokes a relatively small calcium entry in normalHMVECs compared that detected in BTECs and, even moreimportantly, AA is unable to promote cell motility in woundhealing assay in vitro in ECs (21, 22). This opens the intriguingopportunity for differential pharmacological treatments in normaland tumor-derived human ECs.

Fig. 1. Schematic representation of the Ca2� channels involved in the different steps of the tumor vascularization process. Different transient receptor potential(TRP) channels together with Orai1 are widely expressed in endothelial cells (EC) as well as in endothelial progenitor cells (EPC), where they regulates differentprocess involved in tumor vascularization such as proliferation, migration, tubule formation in matrigel, cell reorientation due to shear stress, increase inpermeability, and collateral vessel growth in vivo or in vivo angiogenesis. Numbers in parenthesis indicate the respective reference number. See text for additionaldefinitions of abbreviations.

Themes

C11Ca2� CHANNELS IN TUMOR VASCULARIZATION

AJP-Cell Physiol • doi:10.1152/ajpcell.00280.2011 • www.ajpcell.org

on Decem

ber 28, 2011ajpcell.physiology.org

Dow

nloaded from

A number of cellular stress factors, including hypoxia,nutrient deprivation, and reactive oxygen species, are impor-tant stimuli of angiogenic signaling (55). TRPM2 has beenrecently identified to mediate H2O2-dependent increase inmacrovascular pulmonary EC permeability. TRPM2 knock-down by small interfering RNA (siRNA), inhibition by anti-TRPM2 antibody or overexpression of the TRPM2 short iso-form (that acts as dominant negative for TRPM2 long isoform)significantly reduced the H2O2/Ca2�-mediated increase in ECparacellular permeability (31) (Fig. 1). Finally, although im-plicated primarily in Mg2� rather than Ca2� homeostasis,TRPM7 is involved in a number of vascular pathologies suchas hypertension and dysfunction of endothelial and smoothmuscle cells (73). TRPM7 inhibits human umbilical vein EC(HUVEC) proliferation; transfection of cells with TRPM7-specific siRNA decreased both TRPM7 transcripts andTRPM7-like currents and significantly enhanced HUVEC pro-liferation rate (Fig. 1). The effect is likely due to an increase inERK phosphorylation following the siTRPM7 transfection(36). It is worth noting that TRPM7 has the dual ability to actas a channel and as a kinase, allowing its involvement both incellular Mg2� status and in intracellular signaling.

As briefly described in the previous section of the review,EPCs could be an important component of tumor vascoular-ization, although their role is still debated (24, 59). Interestingdata have been recently published pointing to a role of SOCEin the control of proliferation in EPCs isolated from peripheralblood of human healthy volunteers (65). Suppression of Orai1expression or expression of dominant negative mutants ofOrai1 abolish SOCE in EPCs as well as tubule formation inmatrigel assays (43). Finally, TRPC1 has also been suggestedto contribute to regulation of cell proliferation and cell migra-tion of EPCs isolated from rats bone marrow (40). However,the endogenous stimuli responsible for SOCE activation inEPCs are still unknown.

Macromolecular Complexes and Their Role in Ca2�

Signaling of EC

The specificity of [Ca2�]i signaling is mainly achieved byintracellular molecular complexes, which assemble at precisecellular locations. This localization controls compartmentaliza-tion of Ca2� signals in the cell allowing generation of specificand local signaling events. It is therefore interesting to considerthe localization Ca2� channels in TECs and to determine themilieu in which they function as well as their regulatory andaccessory components. Such information might provide in-sights into their role in tumor angiogenesis.

Protein translocation to the plasma membrane is an impor-tant means to regulate TRP channels. Hormones, growth fac-tors, and agonists targeted for G�q-coupled receptors can stim-ulate the translocation of several TRP channels, includingTRPC1, TRPC3–6, TRPV1, and TRPV4 to the plasma mem-brane (13, 44, 48, 71). As discussed above, AA-induced actinremodeling is associated with TRPV4 trafficking to the plasmamembrane of BTECs, resulting in more functional TRPV4channels in the surface membrane, which promotes Ca2� entryand drives the migration of tumor-derived ECs (23) (Fig. 2).Recently, Ma and co-workers (45) showed that TRPV4-TRPC1 heteromeric channel trafficking to the plasma mem-brane is enhanced by Ca2� store depletion in HUVECs, whichresults in augmentation of [Ca2�]i influx in ECs in response toshear flow. Interestingly, both TRPC1 and TRPV4 subunits arerequested for an efficient membrane translocation (45). InHUVECs, the TRPC1-TRPV4 heteromultimer translocates incaveolar compartments, as indicated by the strong colocaliza-tion with caveolin-1 (45) (see Fig. 2 for a schematic represen-tation). In addition, both TRPV4 and TRPC1 were also previ-ously reported to be colocalized with caveolin-1 in the caveolarcompartment of the plasma membrane in HUVECs andHMVECs, respectively. It is interesting to speculate that inter-

Fig. 2. Schematic representation of the main macromolecular complexes described to have a role in the vascularization process. A: TRPV4 channels colocalizewith actin (1); arachidonic acid (AA)-mediated actin-remodeling promotes TRPV4 vesicle to traffic and insert in the membrane (2); as a consequence morefunctional channels allow Ca2� entry (3) important for tumor-derived endothelial cell (TEC) migration (4). B: vasoactive agonists, acting via specific receptors,induce activation of Ca2� release from the intracellular stores (1, 2), which in turn is sensed by Stim1 and promotes TRPV4-TRPC1 heterotetramers to trafficto the plasma membrane (3) where they localize in caveolin-1-enriched compartments promoting Ca2� entry (4). C: mechanical shear stress applied through theb1 integrins-mediated cell-extracellular matrix adhesions (1) activates TRPV4; consequent Ca2� entry through these channels (2) in turn is required fordownstream signaling events promoting more b1integrin activation (3) that drive the cell and cytoscheletal reorganization response necessary for cell reorientation(4). See text for additional definitions of abbreviations.

Themes

C12 Ca2� CHANNELS IN TUMOR VASCULARIZATION

AJP-Cell Physiol • doi:10.1152/ajpcell.00280.2011 • www.ajpcell.org

on Decem

ber 28, 2011ajpcell.physiology.org

Dow

nloaded from

actions between the caveolar proteins, such as caveolin 1, andTRP channels are required for regulating the Ca2� fluxesthrough the channels and/or regulating the assembly of thechannel complexes in specifc cellular compartments (64, 68).

Caveolae have been described to be involved in the gener-ation of EC plasma membrane signaling microdomains. Cave-olae serve as platforms that sense and transducer variousstimuli, e.g., hemodynamic changes, into intracellular bio-chemical signals that regulate not only Ca2� signaling butdetermine critical stages of growth and angiogenesis both invitro and in vivo (7). Indeed, caveolae are enriched withregulatory molecules like InsP3 receptor, Ca2�-ATPase, het-erotrimeric GTP binding protein, and some TRP channels (61).Specifically, in ECs, caveolar compartments regulate Ca2�

fluxes (both release from intracellular stores and entry) andCa2�-mediated signal transduction (37, 38, 52). Assembly ofthese components into complexes within the caveolar domainsor associated with these domains partitions them into special-ized cellular compartments. Such close association of theproteins provides a route for increasing the specificity and ratesof interactions between them. Scaffolding of effector mole-cules in close vicinity to the complex allows sensing of localsignals. Such spatial compartmentalization is key to a complexprocess such as migration of ECs.

Other important molecular players that have been exten-sively implicated in cancer and vascularization process arethe integrins. Their interaction with channels and functionalroles in pathophysiology have been described earlier and arealso subject of a review by Arcangeli and colleagues (3, 5).Integrins are involved in the Ca2�-mediated vascularizationprocess. In particular, TRPV4 is functionally associatedwith �1-integrins, and stimulation of �1-integrin on ECplasma membrane by mechanical strain promotes TRPV4-mediated Ca2� entry, which in turn activates additional�1-integrins and subsequent downstream cytoskeletal rear-rangement (69) (see also Fig. 2).

Specificity of Proangiogenic Calcium Signaling in Tumor-Derived Endothelial Cells: Concluding Remarks

A number of approaches have been successfully employedto block vascularization in murine tumor models and severalare currently in clinical trials. They are often based on thepharmacological use of blockers specifically targeted to endo-thelial proangiogenic signaling molecules. One of the bestexamples is bevacizumab (Avastatin), an anti-VEGF antibodyand the first antiangiogenic agent approved by the UnitedStates Federal Drug Administration (for colon cancer) (25).The main drawback of this strategy, however, is its poorselectivity being based on biochemical pathways substantiallyshared by normal and tumor-associated vasculature.

Because of its multifaceted role in the control of cell pro-liferation and motility, the Ca2� machinery is a potentialmolecular target for strategies against tumor neovasculariza-tion. Ca2� signals are involved at different critical phases inthe regulation of the complex process of angiogenesis andtumor progression. Carboxyamidotriazole, a nonselective syntheticCa2� influx blocker, has been successfully employed in anti-angiogenic therapy, and it is currently under phase I-III clinicaltrials for the treatment of solid tumors (4, 35). As many cancercells remodel the expression or activity of their Ca2� signaling

toolkit, our group has recently suggested that deregulation ofintracellular Ca2� toolkit might provide a suitable target todesign novel anti-angiogenic therapies (21, 22). On the otherhand membrane channels, as cell surface expressed proteins,are easily and directly accessible via the bloodstream, facili-tating a number of treatment strategies.

However, identifying specific patterns or “fingerprints” ofcalcium channels expression associated with tumor neovascu-larization is a great challenge for basic biology and biomedicalsciences. Since Ca2� signaling encompasses all cell types,developing cell-specific, or even channel-specific, therapies isan extremely difficult problem. In addition, the majority ofendothelial calcium channels have only been detected andinvestigated in cultured EC lines or in primary cultured cells invitro, sparse data are available regarding in situ measurementsin blood vessels. Another important point that needs to beconsidered is that expression of ion channels in ECs criticallydepends on the external conditions (culture passages, growthconditions, confluence) (53). It is therefore not surprising thatthe extensive amount of data on channel physiology in ECs aresometimes variable and quite often controversial and question-able with regard to their physiological significance. A betterapproach in future studies will be to direct efforts to investigatethe candidate channel(s) as well as their physiological functionin vivo. In conclusion, although a challenge for the above-mentioned reasons, Ca2� channels still represent an extremelyuseful, and important, molecular target for intervention oftumor vascularization. Discovery of such therapies and themeans to suppress tumor vascularization will be a milestone inthe quest for cancer therapies.

DISCLOSURES

No conflicts of interest, financial or otherwise, are declared by the author(s).

REFERENCES

1. Abdullaev IF, Bisaillon JM, Potier M, Gonzalez JC, Motiani RK,Trebak M. Stim1 and Orai1 mediate CRAC currents and store-operatedcalcium entry important for endothelial cell proliferation. Circ Res 103:1289–1299, 2008.

2. Antoniotti S, Fattori P, Tomatis C, Pessione E, Munaron L. Arachi-donic acid and calcium signals in human breast tumor-derived endothelialcells: a proteomic study. J Recept Signal Transduct Res 29: 257–265,2009.

3. Arcangeli A. Ion channels in the tumor cell-microenvironment cross talk.Am J Physiol Cell Physiol 301: C253–C254, 2011.

4. Bauer KS, Cude KJ, Dixon SC, Kruger EA, Figg WD. Carboxyamido-triazole inhibits angiogenesis by blocking the calcium-mediated nitric-oxide synthase-vascular endothelial growth factor pathway. J PharmacolExp Ther 292: 31–37, 2000.

5. Becchetti A, Pillozzi S, Morini R, Nesti E, Arcangeli A. New insightsinto the regulation of ion channels by integrins. Int Rev Cell Mol Biol 279:135–190, 2010.

6. Birnbaumer L. The TRPC class of ion channels: a critical review of theirroles in slow, sustained increases in intracellular Ca(2�) concentrations.Annu Rev Pharmacol Toxicol 49: 395–426, 2009.

7. Burgermeister E, Liscovitch M, Rocken C, Schmid RM, Ebert MP.Caveats of caveolin-1 in cancer progression. Cancer Lett 268: 187–201,2008.

8. Bussolati B, Deambrosis I, Russo S, Deregibus MC, Camussi G.Altered angiogenesis and survival in human tumor-derived endothelialcells. FASEB J 17: 1159–1161, 2003.

9. Bussolati B, Deregibus MC, Camussi G. Characterization of molecularand functional alterations of tumor endothelial cells to design anti-angiogenic strategies. Curr Vasc Pharmacol 8: 220–232, 2010.

10. Bussolati B, Grange C, Camussi G. Tumor exploits alternative strategiesto achieve vascularization. FASEB J 25: 2874–2882, 2011.

Themes

C13Ca2� CHANNELS IN TUMOR VASCULARIZATION

AJP-Cell Physiol • doi:10.1152/ajpcell.00280.2011 • www.ajpcell.org

on Decem

ber 28, 2011ajpcell.physiology.org

Dow

nloaded from

11. Cahalan MD. Stimulating store-operated Ca(2�) entry. Nat Cell Biol 11:669–677, 2009.

12. Carmeliet P. Angiogenesis in life, disease and medicine. Nature 438:932–936, 2005.

13. Cayouette S, Boulay G. Intracellular trafficking of TRP channels. CellCalcium 42: 225–232, 2007.

14. Cheng HW, James AF, Foster RR, Hancox JC, Bates DO. VEGFactivates receptor-operated cation channels in human microvascular en-dothelial cells. Arterioscler Thromb Vasc Biol 26: 1768–1776, 2006.

15. Cheng KT, Liu X, Ong HL, Swaim W, Ambudkar IS. Local Ca(2)�entry via Orai1 regulates plasma membrane recruitment of TRPC1 andcontrols cytosolic Ca(2)� signals required for specific cell functions.PLoS Biol 9: e1001025, 2011.

16. De Bock K, Cauwenberghs S, Carmeliet P. Vessel abnormalization:another hallmark of cancer? Molecular mechanisms and therapeutic im-plications. Curr Opin Genet Dev 21: 73–79, 2011.

17. Ebos JM, Lee CR, Kerbel RS. Tumor and host-mediated pathways ofresistance and disease progression in response to antiangiogenic therapy.Clin Cancer Res 15: 5020–5025, 2009.

18. Everaerts W, Nilius B, Owsianik G. The vallinoid transient receptorpotential channel Trpv4: From structure to disease. Prog Biophys Mol Biol103: 2–17, 2009.

19. Fang S, Salven P. Stem cells in tumor angiogenesis. J Mol Cell Cardiol50: 290–295, 2011.

20. Ferrara N, Kerbel RS. Angiogenesis as a therapeutic target. Nature 438:967–974, 2005.

21. Fiorio Pla A, Genova T, Pupo E, Tomatis C, Genazzani A, ZaninettiR, Munaron L. Multiple roles of protein kinase a in arachidonic acid-mediated Ca2� entry and tumor-derived human endothelial cell migration.Mol Cancer Res 8: 1466–1476, 2010.

22. Fiorio Pla A, Grange C, Antoniotti S, Tomatis C, Merlino A, BussolatiB, Munaron L. Arachidonic acid-induced Ca2� entry is involved in earlysteps of tumor angiogenesis. Mol Cancer Res 6: 535–545, 2008.

23. Fiorio Pla A, Ong HL, Cheng KT, Brossa A, Bussolati B, Lockwich T,Paria B, Munaron L, Ambudkar IS. TRPV4 mediates tumor-derivedendothelial cell migration via arachidonic acid-activated actin remodeling.Oncogene, 2011 Jun 20. doi: 10.1038/onc.2011.231.

24. Fischer C, Schneider M, Carmeliet P. Principles and therapeutic impli-cations of angiogenesis, vasculogenesis and arteriogenesis. Handb ExpPharmacol: 157–212, 2006.

25. Folkman J. Angiogenesis. Annu Rev Med 57: 1–18, 2006.26. Fukumura D, Duda DG, Munn LL, Jain RK. Tumor microvasculature

and microenvironment: novel insights through intravital imaging in pre-clinical models. Microcirculation 17: 206–225, 2010.

27. Ge R, Tai Y, Sun Y, Zhou K, Yang S, Cheng T, Zou Q, Shen F, WangY. Critical role of TRPC6 channels in VEGF-mediated angiogenesis.Cancer Lett 283: 43–51, 2009.

28. Grange C, Bussolati B, Bruno S, Fonsato V, Sapino A, Camussi G.Isolation and characterization of human breast tumor-derived endothelialcells. Oncol Rep 15: 381–386, 2006.

29. Hamdollah Zadeh MA, Glass CA, Magnussen A, Hancox JC, BatesDO. VEGF-mediated elevated intracellular calcium and angiogenesis inhuman microvascular endothelial cells in vitro are inhibited by dominantnegative TRPC6. Microcirculation 15: 605–614, 2008.

30. Hartmannsgruber V, Heyken WT, Kacik M, Kaistha A, Grgic I,Harteneck C, Liedtke W, Hoyer J, Kohler R. Arterial response to shearstress critically depends on endothelial TRPV4 expression. PLos One 2:e827, 2007.

31. Hecquet CM, Ahmmed GU, Vogel SM, Malik AB. Role of TRPM2channel in mediating H2O2-induced Ca2� entry and endothelial hyper-permeability. Circ Res 102: 347–355, 2008.

32. Hewett PW. Identification of tumour-induced changes in endothelial cellsurface protein expression: an in vitro model. Int J Biochem Cell Biol 33:325–335, 2001.

33. Hillen F, Griffioen AW. Tumour vascularization: sprouting angiogenesisand beyond. Cancer Metast Rev 26: 489–502, 2007.

34. Hong JH, Li Q, Kim MS, Shin DM, Feske S, Birnbaumer L, ChengKT, Ambudkar IS, Muallem S. Polarized but differential localizationand recruitment of STIM1, Orai1 and TRPC channels in secretory cells.Traffic 12: 232–245, 2011.

35. Hussain MM, Kotz H, Minasian L, Premkumar A, Sarosy G, Reed E,Zhai S, Steinberg SM, Raggio M, Oliver VK, Figg WD, Kohn EC.Phase II trial of carboxyamidotriazole in patients with relapsed epithelialovarian cancer. J Clin Oncol 21: 4356–4363, 2003.

36. Inoue K, Xiong ZG. Silencing TRPM7 promotes growth/proliferationand nitric oxide production of vascular endothelial cells via the ERKpathway. Cardiovasc Res 83: 547–557, 2009.

37. Isshiki M, Anderson RG. Function of caveolae in Ca2� entry andCa2�-dependent signal transduction. Traffic 4: 717–723, 2003.

38. Isshiki M, Mutoh A, Fujita T. Subcortical Ca2� waves sneaking underthe plasma membrane in endothelial cells. Circ Res 95: e11–21, 2004.

39. Jho D, Mehta D, Ahmmed G, Gao XP, Tiruppathi C, Broman M,Malik AB. Angiopoietin-1 opposes VEGF-induced increase in endothelialpermeability by inhibiting TRPC1-dependent Ca2 influx. Circ Res 96:1282–1290, 2005.

40. Kuang CY, Yu Y, Wang K, Qian DH, Den MY, Huang L. Knockdownof transient receptor potential canonical-1 reduces the proliferation andmigration of endothelial progenitor cells. Stem Cells Dev 2011 April 8.

41. Kwan HY, Huang Y, Yao X. TRP channels in endothelial function anddysfunction. Biochim Biophys Acta 1772: 907–914, 2007.

42. Leong S, Dvorak HF, Weaver VM, Tlsty TD, Bergers G. Tumormicroenvironment and progression. J Surg Oncol 103: 468–474, 2011.

43. Li J, Cubbon RM, Wilson LA, Amer MS, McKeown L, Hou B,Majeed Y, Tumova S, Seymour VA, Taylor H, Stacey M, O’Regan D,Foster R, Porter KE, Kearney MT, Beech DJ. Orai1 and CRACChannel Dependence of VEGF-Activated Ca2� Entry and EndothelialTube Formation. Circ Res 108: 1190–1198, 2011.

44. Liu X, Bandyopadhyay BC, Nakamoto T, Singh B, Liedtke W, MelvinJE, Ambudkar I. A role for AQP5 in activation of TRPV4 by hypoto-nicity: concerted involvement of AQP5 and TRPV4 in regulation of cellvolume recovery. J Biol Chem 281: 15485–15495, 2006.

45. Ma X, Cao J, Luo J, Nilius B, Huang Y, Ambudkar IS, Yao X.Depletion of intracellular Ca2� stores stimulates the translocation ofvanilloid transient receptor potential 4-c1 heteromeric channels to theplasma membrane. Arterioscler Thromb Vasc Biol 30: 2249–2255, 2010.

46. Mehta D, Ahmmed GU, Paria BC, Holinstat M, Voyno-YasenetskayaT, Tiruppathi C, Minshall RD, Malik AB. RhoA interaction withinositol 1,4,5-trisphosphate receptor and transient receptor potential chan-nel-1 regulates Ca2� entry. Role in signaling increased endothelial per-meability. J Biol Chem 278: 33492–33500, 2003.

47. Meves H. Arachidonic acid and ion channels: an update. Br J Pharmacol155: 4–16, 2008.

48. Morenilla-Palao C, Planells-Cases R, Garcia-Sanz N, Ferrer-MontielA. Regulated exocytosis contributes to protein kinase C potentiation ofvanilloid receptor activity. J Biol Chem 279: 25665–25672, 2004.

49. Munaron L, Fiorio Pla A. Endothelial calcium machinery and angiogen-esis: understanding physiology to interfere with pathology. Curr MedChem 16: 4691–4703, 2009.

50. Munaron L, Fiorio Pla A. Endothelial calcium machinery and angiogen-esis: understanding physiology to interfere with pathology. Curr MedChem 16: 4691–4703, 2009.

51. Munaron L, Tomatis C, Pla AF. The secret marriage between calciumand tumor angiogenesis. Technol Cancer Res Treat 7: 335–339, 2008.

52. Murata T, Lin MI, Stan RV, Bauer PM, Yu J, Sessa WC. Geneticevidence supporting caveolae microdomain regulation of calcium entry inendothelial cells. J Biol Chem 282: 16631–16643, 2007.

53. Nilius B, Droogmans G. Ion channels and their functional role in vascularendothelium. Physiol Rev 81: 1415–1459, 2001.

54. Nilius B, Owsianik G, Voets T, Peters JA. Transient receptor potentialcation channels in disease. Physiol Rev 87: 165–217, 2007.

55. North S, Moenner M, Bikfalvi A. Recent developments in the regulationof the angiogenic switch by cellular stress factors in tumors. Cancer Lett218: 1–14, 2005.

56. Paria BC, Vogel SM, Ahmmed GU, Alamgir S, Shroff J, Malik AB,Tiruppathi C. Tumor necrosis factor-�-induced TRPC1 expression am-plifies store-operated Ca2� influx and endothelial permeability. Am JPhysiol Lung Cell Mol Physiol 287: L1303–L1313, 2004.

57. Parker BS, Argani P, Cook BP, Liangfeng H, Chartrand SD, ZhangM, Saha S, Bardelli A, Jiang Y, St Martin TB, Nacht M, Teicher BA,Klinger KW, Sukumar S, Madden SL. Alterations in vascular geneexpression in invasive breast carcinoma. Cancer Res 64: 7857–7866,2004.

58. Patton AM, Kassis J, Doong H, Kohn EC. Calcium as a molecular targetin angiogenesis. Curr Pharm Des 9: 543–551, 2003.

59. Pearson JD. Endothelial progenitor cells-hype or hope? J Thromb Hae-most 7: 255–262, 2009.

Themes

C14 Ca2� CHANNELS IN TUMOR VASCULARIZATION

AJP-Cell Physiol • doi:10.1152/ajpcell.00280.2011 • www.ajpcell.org

on Decem

ber 28, 2011ajpcell.physiology.org

Dow

nloaded from

60. Pocock TM, Foster RR, Bates DO. Evidence of a role for TRPCchannels in VEGF-mediated increased vascular permeability in vivo. AmJ Physiol Heart Circ Physiol 286: H1015–H1026, 2004.

61. Remillard CV, Yuan JX. Transient receptor potential channels and caveo-lin-1: good friends in tight spaces. Mol Pharmacol 70: 1151–1154, 2006.

62. Ribatti D. Endogenous inhibitors of angiogenesis: a historical review.Leuk Res 33: 638–644, 2009.

63. Ribatti D. The inefficacy of antiangiogenic therapies. J Angiogenes Res 2:27, 2010.

64. Saliez J, Bouzin C, Rath G, Ghisdal P, Desjardins F, Rezzani R, RodellaLF, Vriens J, Nilius B, Feron O, Balligand JL, Dessy C. Role of caveolarcompartmentation in endothelium-derived hyperpolarizing factor-mediatedrelaxation: Ca2� signals and gap junction function are regulated by caveolinin endothelial cells. Circulation 117: 1065–1074, 2008.

65. Sanchez-Hernandez Y, Laforenza U, Bonetti E, Fontana J, Dragoni S,Russo M, Avelino-Cruz JE, Schinelli S, Testa D, Guerra G, Rosti V,Tanzi F, Moccia F. Store-operated ca(2�) entry is expressed in humanendothelial progenitor cells. Stem Cells Dev 19: 1967–1981, 2010.

66. Sata M. Role of circulating vascular progenitors in angiogenesis, vascularhealing, and pulmonary. Arteriosclerosis 26: 1008–1014, 2006.

67. Seaman S, Stevens J, Yang MY, Logsdon D, Graff-Cherry C, St CroixB. Genes that distinguish physiological and pathological angiogenesis.Cancer Cell 11: 539–554, 2007.

68. Sundivakkam PC, Kwiatek AM, Sharma TT, Minshall RD, Malik AB,Tiruppathi C. Caveolin-1 scaffold domain interacts with TRPC1 andIP3R3 to regulate Ca2� store release-induced Ca2� entry in endothelialcells. Am J Physiol Cell Physiol 296: C403–C413, 2009.

69. Thodeti CK, Matthews B, Ravi A, Mammoto A, Ghosh K, Bracha AL,Ingber DE. TRPV4 channels mediate cyclic strain-induced endothelialcell reorientation through integrin-to-integrin signaling. Circ Res 104:1123–1130, 2009.

70. Troidl C, Troidl K, Schierling W, Cai WJ, Nef H, Mollmann H, KostinS, Schimanski S, Hammer L, Elsasser A, Schmitz-Rixen T, SchaperW. Trpv4 induces collateral vessel growth during regeneration of thearterial circulation. J Cell Mol Med 13: 2613–2621, 2009.

71. Van Buren JJ, Bhat S, Rotello R, Pauza ME, Premkumar LS. Sensi-tization and translocation of TRPV1 by insulin and IGF-I. Mol Pain 1: 17,2005.

72. Yao X, Garland CJ. Recent developments in vascular endothelial celltransient receptor potential channels. Circ Res 97: 853–863, 2005.

73. Yogi A, Callera GE, Antunes TT, Tostes RC, Touyz RM. Transientreceptor potential melastatin 7 (TRPM7) cation channels, magnesium andthe vascular system in hypertension. Circ J 75: 237–245, 2011.

74. Yu PC, Gu SY, Bu JW, Du JL. TRPC1 is essential for in vivoangiogenesis in zebrafish. Circ Res 106: 1221–1232, 2010.

Themes

C15Ca2� CHANNELS IN TUMOR VASCULARIZATION

AJP-Cell Physiol • doi:10.1152/ajpcell.00280.2011 • www.ajpcell.org

on Decem

ber 28, 2011ajpcell.physiology.org

Dow

nloaded from