Targeting Ion Channels in Cancer: a Novel Frontier in Antineoplastic Therapy A

66 Current Medicinal Chemistry, 2009, 16, 66-93 Targeting Ion Channels in Cancer: A Novel Frontier in Antineoplastic Therapy A. Arcangeli*,1, O. Crociani1, E. Lastraioli1, A. Masi1, S. Pillozzi1 and A. Becchetti2

1Department of Experimental Pathology and Oncology, University of Firenze, Italy; 2Department of Biotechnology and Biosciences, University of Milano-Bicocca, Italy Abstract: Targeted therapy is considerably changing the treatment and prognosis of cancer. Progressive understanding of the molecular mechanisms that regulate the establishment and progression of different tumors is leading to ever more specific and efficacious pharmacological approaches. In this picture, ion channels represent an unexpected, but very promising, player. The expression and activity of different channel types mark and regulate specific stages of cancer progression. Their contribution to the neoplastic phenotype ranges from control of cell proliferation and apoptosis, to regulation of invasiveness and metastatic spread. As is being in- creasingly recognized, some of these roles can be attributed to signaling mechanisms independent of ion flow. Evidence is particularly extensive for K+ channels. Their expression is altered in many primary human cancers, especially in early stages, and they frequently exert pleiotropic effects on the neoplastic cell physiology. For instance, by regulating membrane potential they can control Ca2+ fluxes and thus the cell cycle machinery. Their effects on mitosis can also depend on regulation of cell volume, usually in cooperation with chloride channels. However, ion channels are also implicated in late neoplastic stages, by stimulating angiogenesis, mediating the cell-matrix interaction and regulating cell motility. Not surprisingly, the mechanisms of these effects are manifold. For example, intracellular signaling cascades can be triggered when ion channels form protein complexes with other membrane proteins such as integrins or growth factor receptors. Altered channel expression can be exploited for diagnostic purposes or for addressing traceable or cytotoxic compounds to specific neoplastic tissue. What is more, recent evidence indicates that blocking channel activity impairs the growth of some tumors, both in vitro and in vivo. This opens a new field for medicinal chemistry studies, which can avail of the many available tools, such as blocking antibodies, antisense oligonucleotides, small interfering RNAs, peptide toxins and a large variety of small organic compounds. The major drawback of this approach is that some ion channel blockers produce serious side effects, such as cardiac arrhythmias. Therefore, drug developing efforts aimed at producing less harmful compounds are needed and we discuss possible approaches toward this goal. Finally, we propose that a novel therapeutic tactic could be developed by unlocking ion channels from multiprotein membrane signaling complexes.

Keywords: Leukemia, glioma, breast cancer, colorectal cancer, prostate cancer, ion channels, Kv 1.3, KCa 3.1, hEAG-1, Kv 10.1, hERG1, Kv 11.1, Nav 1.5, Nav 1.7.

1. CURRENT VIEWS ON CANCER BIOLOGY AND The molecular dissection of neoplastic progression po- THERAPY tentially opens the way to the development of drugs addressing tumor-specific processes. The many recent efforts de- The molecular biological revolution that has reshaped voted to this task have led to substantial improvement in biomedical research over the past three decades has also treatment. For instance, novel selective inhibitors of receptor changed dramatically our understanding of the origins of and non receptor tyrosine kinases, pivotal regulators of cell neoplasia. The main new notion is probably that cancer cells survival and proliferation, are now available for clinical use. are mutants. They often carry somatic mutations of tumor- An excellent example is the human Epidermal Growth Fac- related genes, although other modifications such as gene tor (EGF) receptor-2 (HER2). Herceptin, the therapeutic amplification or inactivation can also occur, possibly caused monoclonal antibody against HER2 approved by the Food by epigenetic mechanisms. The systematic search for genes and Drug Administration, has been used to treat over particularly liable to mutate during tumour progression has 150,000 women with breast cancer. Considerable advances led to the concept that cancer is a multistep process. Early have also been made in anti-angiogenesis therapy, because steps comprise alteration of a relatively small number of inhibitors of the Vascular Endothelial Growth Factors genes implicated in cell proliferation, apoptosis and differen- (VEGFs) and their receptors revert the tumor-associated tiation. As a consequence, cell clones are produced which are blood vessels to a quasi-normal state. Besides hampering cell invulnerable from apoptosis and capable of unlimited prolif- proliferation, this treatment allows chemotherapeutic drugs eration. The growing tumor mass then stimulates angiogene- to access the tumor’s core. By combining new specific sis, in order to sustain itself. At later stages, phenotypic fea- agents with traditional chemotherapy, patient survival is tures are selected that enable cells to invade and colonize often significantly prolonged. Although many questions still (metastasize) neighbouring or even distant tissue, and even- linger as to the choice of the best drug combination and tually to evade and overcome immune response [1]. regimen calibration, there is little doubt that targeted thera- pies are changing care and prognosis for the better [1].

Here, we review the growing experimental and preclini- *Address correspondence to this author at the Department of Experimental cal evidence indicating that ion channels should be included Pathology and Oncology, University of Firenze, Italy; Tel: +39 055 among the novel targets for cancer therapy, which may open 4598206; Fax: +39 055 4598900; E-mail: [email protected]

0929-8673/09 $55.00+.00 © 2009 Bentham Science Publishers Ltd. Targeting Ion Channels in Cancer Current Medicinal Chemistry, 2009 Vol. 16, No. 1 67 an entire pharmaceutical and clinical field. Why ion chan- the few pharmaceutically tractable molecular classes. A nels? First, their expression is often grossly altered in human major advantage is their accessibility from the extracellular cancers. Second, channel dysfunction can have a strong side, which makes ion channel modulators particularly effec- impact on cell physiology and signaling, with ensuing effects tive. on cancer progression. Third, ion channels represent one of

Fig. (1). Dendrogram of the different families of K+ Channels, Voltage-Gated Ca2+ Channels (VGCC) and Voltage-Gated Na+ Chan- + + + nels (VGSC). For K Channels, the four main families are shown: Voltage-Gated K Channels (VGKC or Kv ), Inwardly-Rectifying K 2+ + + Channels (KIR), Ca -activated K Channels (KCa ) and Two-pore K Channels (K2p). Subtypes are named according to the IUPHAR nomenclature. The channel types mainly expressed in cancer cells are highlighted. Box: comparison of IUPHAR, HGNC and common names for the K+ channels discussed in the text and tables. 68 Current Medicinal Chemistry, 2009 Vol. 16, No. 1 Arcangeli et al.

2. ION CHANNEL STRUCTURE AND PHYSIOLOGY: Finally, the Transient Receptor Potential (TRP) gene su- A BRIEF SURVEY perfamily is also related to the VGC’s, thus retaining the structural features outlined above. Subunits form homo- or Ion channels are integral membrane proteins that control heterotetrameric channels. Six TRP families are known passive ion fluxes, by switching between non conductive (TRPC, TRPV, TRPM, TRPA, TRPP and TRPML), with (‘closed’) and conductive (‘open’) conformational states. different permeability ratios between Ca2+ and monovalent Such a ‘gating’ process (activation) is often driven by trans- cations, ranging from nil to very high. These proteins are membrane voltage (Vm) or specific ligands, although other widely distributed in mammalian tissues and have been im- mechanisms are possible. Many types of ion channels exist, plicated in several human diseases, including cancer. The with widely different degrees of ion selectivity and kinetic physiological meaning of altered TRP channel expression in features, which produces great functional flexibility. Volt- tumors is under intense study [8]. age-gated channels (VGCs) usually regulate cellular excit- Fig. (1) shows a dendrogram of VGCs with the corre- ability and shape the action potential. In addition, by control- 2+ sponding IUPHAR nomenclature. Channels that have been ling transmembrane Ca fluxes, they can trigger exocytosis detected in cancer cells are highlighted. For the K+ channels and muscle contraction. Ligand-gated channels typically discussed in the text, both the IUPHAR and the common exert synaptic roles. The integrated action of different channel types controls cell volume, transepithelial ion flow, sen- names are given (box). sory transduction, synaptic function and many other physio- The other side of the ion transport coin is constituted by logical processes. ion pumps and other transporters. Knowledge about the transporters’ role in neoplasia is however less extensive than If the stimulus is interrupted, channels close (deactiva- it is for ion channels. Therefore, we will limit our discussion tion). Otherwise, they often enter a further non conductive + + + state, by a process named inactivation for voltage-gated to some specific issues concerning H fluxes and the Na /K pump, omitting a detailed treatment of the structure and channels and desensitization for the other channel types. function of these proteins. Cells can regulate the energy difference between states and the kinetics of the transition processes, for example by allos- teric regulators or phosphorylation. 3. DIFFERENT CHANNELS IN DIFFERENT TU- MORS From a structural point of view, ion channels are usually composed of different subunits or domains that surround one Numerous reviews have described what is known about or, less commonly, more conduction pathways. We briefly the role of specific channel types in cell proliferation and discuss the VGC family, which comprises most of the ion tumor progression [9-16]. A complementary standpoint con- channels we mention in the main text [2, 3]. Voltage-gated + siders the full ion channel complement of different tumors K channels (VGKCs or Kv) and the related cyclic nucleo- [14]. This is a premise to an integrated pathological interpre- tide-gated channels are formed by four subunits surrounding tation for different neoplasias and allows to discern general a central pore. An example is given in Fig. (4). Each subunit patterns. We adopt the latter approach, although the picture contains six transmembrane segments (S1-S6). Both the N- is far from being complete. Many studies have addressed and the C-termini are intracellular. S5 and S6 are connected transformed cell lines, rather than primary cultures from by a pore loop that contributes to both ion selectivity and surgical samples. Moreover, most data concern a few com- gating [4-6]. The intracellular domains contain consensus mon tumors. The following survey updates the reader about sequences for phosphorylation and the N-terminus deter- the expression (Table 1) and the putative role (Table 2) of mines interaction with other subunits or regulatory proteins. + 2+ individual ionic currents in the indicated tumors. More his- The above pattern is also shared by Na and Ca channels, torical and mechanistic details are given for blood and brain except that the four elements that surround the pore are not cells, because long course studies in these tissues have set independent subunits, but are repeated domains of a continu- + the stage for some current lines of the field. For the other ous polypeptide. Each domain is homologous to a K chan- tumors, treatment is more schematic and full introduction to nel subunit. VGCs have intrinsic voltage-dependency, i.e. literature is found in the Tables. These also summarize what their conformation is controlled by Vm, whereas auxiliary is known on the expression and function of some ion pumps proteins ( subunits) modulate accessory properties. An in neoplastic cells. array of genes coding for mammalian VGCs are known, to which a standard nomenclature has been attributed. When a. Leukemia/Lymphoma describing cellular currents, it is however still customary to use classic names, which define broad functional features Lymphocytes common to different gene products. For example, many K+ Work carried out in the early eighties led to the discovery channels belonging to the K 1 and K 10 molecular families v v of ion channels in lymphocytes and suggested specific chan- produce delayed outward rectifying currents that activate on nel roles in lymphocyte activation and function [17-23]. In depolarization and usually determine the repolarization particular, work done in M. Cahalan’s research group indi- phase of the action potential. These channels are collectively cated that VGKCs could regulate mitogenesis, in T cells. named K . The inward rectifying K+ (IRK) channels are DR Subsequent work from this and other groups aimed at clari- evolutionary related to the VGC family. They are also + fying the differential expression of K channels in T lym- formed by four subunits, each containing only two trans- phocyte populations and how they control T cell activation membrane domains (M1 and M2), homologous to the S5 and [24-27]. These cells turned out to express delayed rectifying S6 of VGCs and connected by a pore loop [7]. Targeting Ion Channels in Cancer Current Medicinal Chemistry, 2009 Vol. 16, No. 1 69

Table 1. Summary of the Ion Channels Expressed in Cell Lines and/or Primary Tumors Discussed in this Article

Ion channel type Evidence of altered expression References

Leukemia/Lymphoma: Cell lines Myeloblastic leukemia 4-AP sensitive K+ channels [32-35]

Monocytic leukemia KDR [43]

Basophilic leukemia KIR; CRAC [36]

Erythroleukemia KV 11.1; KCa 1.1 [38-41]

Acute myeloid leukemia KV 11.1, KV 1.3 [44-46]

T leukemia KV 1.3, KCa 2.2; NaV 1.5 [225-228]

B lymphoma KV 1.3; KV 11.1 [37,44]

KCa 3.1 Increased expression by serum addition [37]

B-ALL KV 11.1; KV 12.2 [44,47] Primary Tumors

AML KV 11.1 ; presence of splice variants [45,46]

B-ALL KV 11.1 [47]

DLBCL (germinal center) KCa 2.3 [48]

DLBCL (activated B-like) KV 1.3 [48] Glioma: Cell lines

KCa 1.1 Stimulated by GF/neurotrasmitters [60]

ClC 2-3-5; Deg/ENaC member; KIR 2.1; KV 11.1; [59,61,64, KV 12.2; TRPC 1-3-4-5-6; TRPM8 65, 70, 71] + + Na /K ATPase 1 subunit [213] Primary Tumors Astrocytomas KIR 2.1, KIR 4.1, KV 1.5, KV 10.1, KV 11.1, [57, 59, 65; NaV 1.1-1.2-1.3-1.6-2.1, KV 12.2, TRPC1-3-4-5-6 67-70] Na+/K+ ATPase 1 subunit [213]

Oligodendrogliomas KIR 2.1, KV 11.1, KV 1.5, KV 10.1, NaV 1.1-1.2-1.3 [65, 67, 71] Breast cancer:

Cell lines NaV 1.5; NaV 1.5; NaV 1.5; CaV 3.2; KV 1.1, [72-79, 83, 104] KV 10.1, KV 11.1;KCa 3.1; BK(L) K2p 9.1 Genomic amplification and over- expres- [83] sion in primary tumors VGCC [110] SOC Activation by heregulin through c-erbB2 [74] CLCA2 [90] Primary Tumors NaV 1.5 [76] CaV 3.2; KIR 3.1, K2p 9.1, KV 1.3, [73, 80-85] CLIC4 [86] CLCA2 Loss of expression due to hypomethylation [90] of the promoter

Prostate cancer:

Cell lines VGCC L-type, TRPC1-4-6, TRPM8, TRPV6, NaV [110, 92-95, 97- 1.7; 99] K2p 2.1 BK (L) Regulated by serum-derived factors [106] KCa 1.1 Genomic amplification (in late stage tu- [104] VGSC mors) [105] Primary Tumors TRPM8,TRPV6, NaV 1.7; Increased expression by EGF [100,101] K2p 2.1 [95, 96, 98, 99]

KV 1.3 [106]

KCa 1.1 [103] KCNRG Genomic amplification (in late stage tu- [105] mors) [107] Putative tumor suppressor

70 Current Medicinal Chemistry, 2009 Vol. 16, No. 1 Arcangeli et al.

(Table 1). Contd…..

Ion channel type Evidence of altered expression References

Colon cancer: Cell lines VGKC [108]

KV 1.3, KV 1.5, KV 3.1, KV 3.4, KV 11.1 [109, 112, 113]

KV 10.1 Genomic amplification [112] VGCC [110] SOC [229]

Primary Tumors KV 1.3, KV 1.5, KV 3.1, KV 10.1, KV 11.1, [112, 113]

K2p 9.1 Genomic amplification [114]

KV 7.5 Genomic mutation [116] CLCA1, CLCA2 [118] + + Na /K ATPase 1 subunit, 2 subunit [213] Gastric cancer:

Cell lines KV 11.1, KV 10.1 [119-122] H+/K+ pump [125]

Primary Tumors KV 11.1, KV 10.1 [119-122]

KV 7.1 Mutations in mouse models predispose to [123, 124] metaplastic and neoplastic changes.

Lung cancer: Cell lines SMCLC VGSC, GIRK1-2-3-4 [128, 129, 127, NSCLC nAChRs, VGSC, GIRK1-2-3-4 230] + + Na /K ATPase 1 subunit [126, 127, 130, 230]

[213] Primary tumors nAChRs + + NSCLC Na /K ATPase 1 subunit [126] [213] Neuroblastoma:

Cell lines KDR [231]

KV 11.1 Expression of splice variants [137]

KV 10.1 [231]

Primary tumors KV 11.1 , expression of splice variants [138]

NaV 1.7 [232] The first column (Ion channel type) shows the ion channel types found to be expressed in the different tumor cells and tissues. The IUPHAR nomenclature is used when the channel type is defined, otherwise the common current name is given. The second column (Evidence of altered expression) shows the mechanisms (genetic or not) leading to ion channel altered expression in tumor cells compared to the normal ones. When such mechanisms have not been clarified and quantified, no indication is shown. : increased expression; : decreased expression; VGCC: voltage gated calcium channels; VGSC: voltage gated sodium channels; CRAC : Ca2+ release-activated Ca2+ channels; SOC: store-operated Ca2+ channels; nAChRs: nicotinic acetylcholine receptor; NSCLC: non small cell lung carcinoma; SCLC: small cell lung carcinoma. References are given in brackets. + 2+ K channels (Kv 1.3) and intermediate conductance Ca - arrest in G1, with no evidence of cell differentiation [33]. + + + dependent K channels (KCa 3.1) [28-30]. The K channel- Therefore, K channels in ML-1 cells appear to be strictly dependent hyperpolarization facilitates the Ca2+ influx in- linked to the cell cycle control. Consistently, K+ currents are duced by antigen binding. The consequent stimulation of inhibited when cells are arrested in G1 by serum deprivation, intracellular Ca2+- and PKC-dependent pathways triggers and restored on serum re-addition or EGF application [34]. proliferation (reviewed in [31]). The process depends on channel phosphorylation [35]. Leukemia and Lymphoma: Cell Lines The possible effects of K+ channel activation on Ca2+ + fluxes have not been tested in ML-1 cells, but were demon- A similar scheme may apply to transformed cell lines. K currents seem often to be necessary during proliferation, strated in a rat basophilic leukemia cell line, RBL-1, which expresses IRK channels. These probably maintain a favour- although which kind of channel is involved depends on cell able driving force for Ca2+ influx through store-operated type. Early evidence was obtained in the myeloblastic leu- Ca2+ release-activated Ca2+ (CRAC) channels [36], in kemia cell line ML-1. When proliferating, these cells express + agreement with the early hypothesis based on work in T functional K channels sensitive to 4-amino-pyridine (4-AP), cells. In other cases, the relation between K+ channels and which are instead suppressed after inducing macrophage 2+ 2+ differentiation [32]. Treatment with 4-AP makes ML-1 cells Ca flux is more complex, with Ca producing feed-back on K+ currents themselves. For example, the human Daudi Targeting Ion Channels in Cancer Current Medicinal Chemistry, 2009 Vol. 16, No. 1 71

Table 2. Ion Channel Roles in Cancer Progression Discussed in this Review

Role in tumor Ion channel type Mechanism of action References progression

a) Cell prolif- K+ channels 4-AP sensitive K+ channel Modulation of signalling pathways [32-35, 167] eration K 1.3, K 10.1 Control of V and of [Ca2+] Modulation of [78, 85, 109] v v m I intracellular pH [109]

Kv 1.5, Kv 3.1, Kv 3.4 Modulation of intracellular pH. Control of Vm [109] 2+ and of [Ca ]I

Kv 11.1 Formation of molecular complexes. Modulation [46,47,65,177] of signalling pathways.

- KCa 1.1 Control of cell volume in concert with Cl chan- [60,105] nels

2+ KCa 3.1 Interplay with Ca influx [78,234]

K2p 2.1 [106]

2+ VGCC Cav 3.2 Control of [Ca ]i [72] Cation channels: ASIC1 Control of cell volume [64] Deg/ENaC

2+ SOC Control of cell prolifera- Control of [Ca ]i [62] tion TRP TRPC-1, TRPC-3, TRPC- Modulation of Ca2+ entry [71][94] 5, TRPC-6, TRPV6 Ligand gated ion channels nAChR Activation of ERK and PI3-k/mTOR pathways [128] Ion Transporters Na+/K+ ATPase [213] (V)-ATPase [149, 150]

b) Cell invasion VGKC Kv 11.1 Formation of molecular complexes. Modulation [46, 113] of signalling pathways

Nav 1.5 Increase of cysteine cathepsin activity [63]

VGSC + Nav 1.7 Na influx and subsequent PKA activation [97-99, 232] Cation channels: ASIC1 Control of cell volume [64] Deg/ENaC Ligand gated ion channels nAChR Activation of ERK and PI3-k/mTOR pathways [128]

2+ c) Differentia- KCa Control of oscillatory Ca signalling [42] tion

d) Apoptosis VGKC Kv 11.1 Regulation of TNF-induced apoptosis [235]

Kv 1.5 Apoptosis resistance due to low expression [236] TRPC1, TRPC4, TRPM8 Ca2+ entry.Support to the androgen-dependent [94,95] 2+ TRP component of store-operated Ca entry

- e) Control of KCa KCa 1.1 Control of cell volume in concert with Cl [60] cell volume channels KCNRG [107] Cl- channels ClC-3 Control of cell volume in concert with BK [59] channels Ca2+ activated- chloride CLCA1, CLCA2 [118] channels

72 Current Medicinal Chemistry, 2009 Vol. 16, No. 1 Arcangeli et al.

(Table 2). Contd…..

Role in tumor Ion channel type Mechanism of action References progression

f) Correlation Kv 1.3 Poor outcome (prostate) [103] with clinical- pathological Kv 10.1 Adverse prognosis (colon) [112] parameters and VGKC Kv 11.1 Shorter overall survival (leukemias). Tumor [46,65,113] prognosis in grade (gliomas). Adverse prognosis (colon) primary sam-

ples IRK channels KIR 4.1 Tumor grade (gliomas) [59, 66]

KIR 3.1 Lymph node involvement (breast ) [80]

+ Two-pore K channels K2p 2.1 Tumor grade (prostate) [106

VGSC Nav 1.7 Potential diagnostic marker (prostate) [99] VGCC L-type Involvement in cancer development and progres- [99] sion mediated by androgen (prostate) TRP TRPV6 Correlation with Gleason score (prostate) [94]

The first column shows the main cellular functions altered during tumor progression. The second column shows the ion channel types expressed in the different tumor cells and tissues. The IUPHAR nomenclature is used when the channel type is defined, otherwise the common current name is given. The third column shows the cellular mechanisms mediating ion channel functions in the tumor progression steps indicated in the first column. When such mechanisms have not been clarified and quantified, no indication is shown. Refer- ences are given in brackets. : Voltage Gated Potassium Channels; KCa: Calcium-activated Potassium Channels; VGCC: voltage gated calcium channels; VGSC: voltage gated sodium channels; ASIC1: acid-sensing ion channel 1; IRK: inwardly rectifying K+ channels; nAChRs: nicotinic acetylcholine receptor. SOC: Store Operated Potassium Channels; TRP: Transient Receptor Potential Channels. cell line, a model of B-lymphoma, expresses functional Kv 11.1 channels (hERG1 currents) led cells to pause in G1 1.3 and KCa 3.1. Specific block of KCa 3.1 inhibits cell cycle, [45]. What is more, KV 11.1 expression was correlated with a whereas the opposite occurs when these channels are up- more aggressive AML phenotype both in vitro and in vivo. In regulated by serum addition [37]. Further details are given in a cohort of patients affected by AML, Kv 11.1 expression Table 1. was associated with a higher probability of relapse and a shorter overall survival [46]. This is one of the first clinical The regulatory complexity is considerably increased by and prognostic applications of an expression screening for a the fact that, in other contexts, the effects of K+ channels VGKC (see Table 2). Similar results were obtained in child- directly modulate cell differentiation, instead of cell cycle. hood B-acute lymphoblastic leukemia (B-ALL) [47]. Both B- This was formerly observed in Friend erythroleukemia cells ALL cell lines and primary B-ALL cells expressed func- (MELC), which express Ca2+-dependent K+ channels (BK ) Ca tional K 11.1 channels, and K 11.1 inhibition impeded the [38,39]. These are transiently activated when differentiation v v bone-marrow induced protection against chemotherapeutic is stimulated by cell adhesion onto fibronectin [40,41], or by drugs, thus restoring a substantial apoptotic cell death. The application of classical inducers of erythroid differentiation K 11.1 role in cancer cell biology is thus very complex. [42]. Similar effects were observed in THP-1 human mono- v Mechanistic hypotheses based on current evidence are dis- cytic leukemia cells. Undifferentiated THP-1 cells express cussed later. KDR channels. When differentiation to macrophages is induced by phorbol esters, KDR expression is turned off, The gene expression pattern of some primary tumors has whereas BKCa and IRK are turned on [43]. A full discussion also been profiled with DNA microarrays. These studies of the K+ channel effects on differentiation is outside the often report altered expression of ion channels and transport- scope of the present review. We limit ourselves to exhort the ers. For example Alizadeh and colleagues (see Fig. 4 in [48]) reader to keep in mind the possible complementary effects found that KCNN3 (KCa 2.3) was up-regulated in germinal exerted by channel modulation on the proliferation and center B-like diffuse large B cell lymphoma (DLBCL), differentiation branches of cell signaling. whereas KCNA3 (Kv 1.3) was up-regulated in activated B- Leukemia and Lymphoma: Primary Tumors like DLBCL. + An extensive study of the K channel transcripts in pri- b. Brain Tumors mary lymphocytes and leukemias as well as several hematopoietic cell lines has been carried out by Smith and col- Unlike other cancers, glial tumors (i.e. gliomas, compris- leagues [44]. In particular, they tested Kv 1.3, Kv 10.1, Kv 11.1 ing astrocytomas and ologodendrogliomas) do not produce and Kv 12.2. Among these, only Kv 11.1 (hERG1) turned out metastases outside the central nervous system by spreading to be up-regulated in cancer cells. Expression was however through blood. Instead, they invade the brain and spinal cord not related to proliferation per se, because it was not ob- by active cell migration. In order to effectively navigate the served in proliferating noncancerous lymphocyte types such tortuous and narrow extracellular spaces, glioma cells need as activated tonsillar cells, lymphocytes from Sjögren's pa- to control their shape and volume in a sophisticated way. To tients and Epstein-Barr virus-transformed B cells. Con- this purpose, they develop a cellular apparatus devoted to versely, we have found Kv 11.1 transcript and the corre- achieve a precise modulation of ion and water fluxes. Be- sponding currents in acute myeloid leukemia (AML) cell cause this process is mixed with the normal channel contri- lines and in a high percentage of primary blasts from AML bution to proliferation, the expression and physiology of ion patients. In this case, the block of current carried through Kv channels in gliomas is particularly intricate. Targeting Ion Channels in Cancer Current Medicinal Chemistry, 2009 Vol. 16, No. 1 73

Normal Glia sion of the TRPC channel clones TRPC-1, TRPC-3, TRPC-5 and TRPC-6 has also been observed in gliomas. This is in- Glial cells have been observed, somewhat unexpectedly, teresting from our perspective because chronic application of to express a variety of VGCs, whose function is still matter the TRPC inhibitor SKF96365 causes near complete growth of debate (reviewed in [49, 50]). From our standpoint it is important to recall that a shift in the K+ channel expression arrest [71]. occurs during astrocyte development [51-53]. During immature stages, cycling glial cells express delayed and transient c. Breast Cancer outward VGKCs and are relatively depolarized, with Vm Ca2+ Channels around -50 mV. Mature quiescent astrocytes have instead a Most of the studies concern human breast carcinoma cell rather hyperpolarized Vm (about -80 mV), which recent work lines, which express voltage gated Ca2+ currents (VGCCs), mainly attributes to the expression of KIR 4.1 IRK channels [54, 55]. This transition correlates with inhibition of cell mainly of the T-type [72]. Expression of the gene encoding CACNA1H (Ca 3.2) has been detected in both breast cancer proliferation [56]. Moreover, functional expression of KIR v cell lines and specimens from primary breast cancers [73]. 4.1 in astrocytomas tends to block proliferation, a process 2+ impeded when these currents are inhibited [57]. A similar Breast cancer cells also express store-operated Ca channels (SOCs), seemingly stimulated by the oncogenic protein c- pattern is observed in oligodendrocytes, where KDR currents have been recorded in proliferating, immature cell, whereas a erbB2 [74]. reduction in the expression of the corresponding channels Na+ Channels (Kv 1.2 and Kv 1.5) accompanies maturation and exit from cell cycle [58]. The highly invasive MDA-MB-231 cell line expresses voltage gated Na+ channels (VGSC), particularly a neonatal Gliomas form (Nav 1.5), whose inhibition impairs cell motility [75- An intricate network of ion channel expression and func- 77]. Importantly, Nav 1.5 expression is also observed in vivo tion has been highlighted by the detailed studies carried out [76]. The activity of Nav 1.5 is thought to stimulate invasive- in H. Sontheimer’s laboratory, on both glioma cell lines and ness through increased cysteine cathepsin activity [63]. primary samples [59-61]. Cell shrinkage is initiated by efflux K+ Channels of KCl through activation of Cl- and K+ channels, with water following osmotically through aquaporins. The main K+ Extensive data have been collected from the human channel expressed by gliomas is gBK, encoded by a splice MCF-7 cells (reviewed in [78]). These express VGKC (Kv 1.1 and Kv 10.1) and KCa 3.1. Inhibiting Kv 10.1 (EAG-1) and variant of the hslo BK channel gene (KCNMA1 or KCa 1.1). gBK is highly sensitive to changes in cytosolic Ca2+ and can KCa 3.1 leads to cell accumulation in G1. It has been pro- be activated by growth factors and neurotransmitters. Ca2+ posed that Kv 10.1 allows entry into G1, and then KCa 3.1 influx through Ca2+ permeable alpha-amino-3-hydroxy-5- drives progression through G1 and into S phase. Consis- methyl-4-isoxazolepropionic acid (AMPA) receptors is be- tently, growth factors (GFs), such as insulin-like growth lieved to be a major physiological stimulus [60]. The main factor 1 (IGF1), increase the expression of both Kv 10.1 and Cl- channel involved in glioma cell shrinkage appears to be KCa 3.1 and induce cell proliferation. Kv 11.1 is also ex- ClC-3, a channel typically found in endocytic vescicles [59]. pressed in MCF-7 cells, but probably contributes to regulate Inhibitors of either gBK or Cl- channels potently block cell volume and not proliferation, as suggested by applica- glioma cell invasion, in vitro and in situ. Cl- channels, in tion of specific inhibitors [79]. particular, are effectively inhibited by chlorotoxin (Cltx), a Studies in primary human breast cancers revealed over- scorpion derived peptide that, upon binding, induces ClC-3 expression of the G-protein-modulated inwardly rectifying - endocytosis into caveolae. This causes Cl channel depletion potassium channels (GIRK), mainly GIRK1 (KIR3.1), which from the plasma membrane, which impairs the cell capacity correlated with metastatic density in lymph nodes [80-82]. to regulate its volume [62,63]. Finally, glioma cells also Subsequently, KCNK9 (a two-pore TWIK-Related Acid + express an amiloride- and psalmotoxin-sensitive cation cur- sensitive K channel, K2p 9.1) was also found to be overex- rent that is not found in normal human astrocytes [64]. This pressed in these tumors, because of strong gene amplification current type has been proposed to contribute to the regula- [83]. Basing on experimental overexpression in murine fi- tory volume increase that restores cell volume during both broblasts, it has been proposed that KCNK9 confers high cell cycle and the glioma cell migration within the cerebral resistance to hypoxia and serum deprivation [84]. It should interstices. also be mentioned that an ample survey of human breast However, as in the case of blood cells, the expression cancer specimens showed a positive immunoreaction for Kv pattern and physiology of K+ channels in gliomas is turning 1.3 in all the examined samples [85]. out to be much more complex than previously suspected. Cl- Channels Table 1 and Table 2 detail, respectively, the expression and A member of the intracellular chloride channel (CLIC) function of different channel types in glioma cell lines and in family, CLIC4, has been suggested to be a tumor suppressor. primary cultures obtained from surgical specimens of patients affected by astrocytomas of different grade. The ex- Loss of CLIC4 in tumor cells with corresponding increase in + + stroma is common in human breast cancers and marks ma- pression of different K channels, Na channels and TRPM8 lignant progression [86]. A different role of CLIC4 has been has been observed in gliomas and sometimes correlated with reported in squamous and mesenchymal (osteosarcoma) tumour grade [65-70]. A brief discussion of this complexity cancers [87]. In fact, CLIC4 expression increases in kerati- is deferred to paragraph 5 and 6. Finally, significant expres- 74 Current Medicinal Chemistry, 2009 Vol. 16, No. 1 Arcangeli et al. nocytes, after exposure to DNA damaging, cancerogenic, pattern and function was recently attributed to the K2p chan- agents and CLIC4 reduction through antisense oligonucleo- nel TREK-1 (K2p 2.1) [106]. Finally, a putative prostate tides causes apoptosis [87]. cancer tumor suppressor gene has been identified in the KCNRG gene, which maps on chromosome 13q14.3 and The CLCA protein family also merits a brief mention. CLCA’s have high homology to cell adhesion proteins and encodes for a protein with high homology to the tetrameriza- tion domain of VGKCs [107]. have been implicated in metastatic processes, although it is still uncertain whether they form Cl- channels, or are accessory subunits [88]. CLCA2 is often lacking in breast cancers e. Colon Cancer and appears to be an excellent candidate for the 1p31 breast K+ Channels cancer tumor suppressor gene [89,90]. Moreover, in endothelial cells, CLCA2 behaves as a vascular addressin for metas- VGKCs appear to exert a pleiotropic role in regulating tatic, blood-borne, cancer cells. It thus facilitates vascular colorectal cancer cell proliferation and progression. The first report was provided by Yao and Kwan [108], who showed arrest of cancer cells via adhesion to 4 integrins, and hence + promotes early metastatic growth [91]. that K channel inhibitors reduce cell proliferation in the colon carcinoma cell line DLD-1. Subsequently, different d. Prostate Cancer VGKCs were detected in the colon carcinoma cells T84, such as Kv 10.1, Kv 3.4 and Kv 1.5 [109]. Application of Ca2+ Channels channel inhibitors as well as specific small interfering (si) Expression of VGCC (mainly L-type) has been shown in RNAs led to conclude that Kv channels control proliferation, 2+ in these cells. Moreover, K 1.3, K 1.5, K 3.1, K 10.1 [110- the androgen-responsive LNCaP cells, in which Ca cur- v v v v 112], K 11.1 [113] and K 9.1 [114] transcripts have been rents are activated by androgens and mediate the androgen- v 2p 2+ detected in primary human samples of colon carcinoma. induced effects [92]. Part of the Ca effects must depend on + These results agree with the observation that K genes are up- stimulation of K channels, as blocking intermediate conduc- v regulated in the colon of mice treated with chemical carcino- tance KCa inhibits the proliferation of prostatic cancer cells [93]. Ca2+ influx through TRPCs also occurs and pro- gens [112]. The oncologic relevance turns on the fact that genomic amplification of K 10.1 is an independent marker motes either cell proliferation or apoptosis, depending on V of adverse prognosis [112] and that Lastraioli et al. [113] TRPC subtype (see Table 2) [94]. TRPM8 is especially in- found a high correlation between the level of K 11.1 surface teresting, because its expression and subcellular distribution v expression and carcinoma stage. Moreover, a negative corre- are regulated by androgens and correlated to metastatic po- 2+ lation was observed between K 11.1 expression and tumor tential. The TRPM8-dependent increase in cytoplasmic Ca v could contribute to the development of androgen independ- chemosensitivity to doxorubicin [115]. ence [95]. TRPV6, finally, is absent in the healthy prostate Recent work has investigated which genes are mutated at and benign prostatic hyperplasia, but is highly expressed in significant frequency, in a subset of human colorectal cancer prostatic cancer specimens, with a significant correlation samples. Kcnq5 (Kv 7.5) turned out to be frequently mutated with the Gleason score [96]. [116], whereas Scn3b (codifying for the subunit of the type + Na+ Channels III VGSC) and Kctd15 (K channel tetramerisation domain 15) were among the genes synergistically controlled by the Work of M.B. Djamgoz and colleagues has shown that in mutant p53 and Kras, typical oncogenes of murine and hu- prostatic cancer, in vitro, Nav 1.7 expression is associated man colon cancers [117]. with a strong metastatic potential and its activity potentiates Cl- Channel-Related Proteins cell migration, crucial for the metastatic cascade [97,98]. This and other VGSC -subunits are also detected in normal In a cohort of patients affected by colorectal carcinoma, prostatic tissue, but at a much lower level. Nav 1.7, in par- CLCA1 and CLCA2 showed widespread downregulation ticular, could be a useful diagnostic marker [99]. [118]. In normal colon epithelium, the mRNA levels of CLCA1 correlated with c-myc transcription, but this coupling VGSC up-regulation in strongly metastatic human and rat disappeared in the tumor samples. Transcription of both prostatic cancer is dependent on EGF [100, 101]. However, in the strongly metastatic Mat-LyLu model of rat prostate genes was barely detectable in different colorectal cancer cell lines. Therefore CLCA proteins, in analogy with breast cancer, VGSC expression shows auto-regulation, through + cancer, could be tumor suppressor in colorectal cancer as activation of protein kinase A (PKA) stimulated by Na well. influx itself [102]. + K Channels f. Gastric Cancer Kv 1.3 is mainly expressed in early stages of progression K+ Channels and down-regulated in high grade cancers [103]. As in the case of other tumors, BKCa channels have also been detected: Only sparse information is available. Shao et al. showed the novel BK(L), whose expression is independent from the that Kv 11.1 is expressed in gastric cancer cell lines and pri- androgen level [104], and KCa 1.1, whose gene, located in mary tumors and that the hERG1 blocker cisapride inhibits 10q22 chromosome, is amplified in late-stage human pros- cell cycle [119,120]. Consistently, we have found Kv 11.1 in tate cancers. Overexpression of KCNMA1 regulates cell pre-cancerous lesions of the esophageal lower tract and proliferation, as witnessed by the growth inhibiting effect of stomach, with high correlation with later progression to- the specific blocker iberiotoxin [105]. A similar expression wards adenocarcinomas. The Kv 11.1 membrane expression Targeting Ion Channels in Cancer Current Medicinal Chemistry, 2009 Vol. 16, No. 1 75 and activity modulates secretion of VEGF-A, probably sion is in broad agreement with current genetic evidence, through transcriptional regulation [121]. The Kv 10.1 tran- because no clear cancer-related mutation in any channel- script and protein were also detected in gastric cancers and encoding gene has been so far reported. Therefore, it would associated with cancer lymph node metastasis and stage appear that ion channels are not so much involved in tumor [122]. Finally, mice carrying mutant alleles of kcnq1 in a causation, but are implicated in the different stages of neo- pathogen-exposed environment rapidly develop gastritis, plastic progression. A partial exception is KCNRG, which metaplasia, dysplasia and pre-malignant adenomatous hy- encodes a K+ channel-regulating protein that has been pro- perplasia [123,124]. posed to be a tumor suppressor gene [107]. A missense mutation at the codon 92 of KCNRG is often present in human Ion Transporters hepatocellular carcinomas, positive for the Hepatitis B virus Because of the importance of the H+/K+ pump in control- [132]. Another partial evidence is the observation that a ling the gastric pH homeostasis, we briefly mention that Kv 11.1 variant appears to predispose to the development of induction of this transporter accompanies the neoplastic the benign tumors leading to the Conn’s syndrome [133]. transformation of a gastric cell line by chemical mutagenesis. The frequent overexpression of channel-encoding genes Although this points to some degree of correlation with gas- + + in human cancers seems to be often caused by gene amplifi- tric cancer, the exact involvement of H /K pump in the acquisition of tumor-like phenotype is still unclear [125]. cation. This has been demonstrated for KCNK9, in breast [83] and colorectal cancers [114], and CACNA1E (Cav 2.3), in Wilms’ tumours [134]. g. Lung Cancer In other cases, epigenetic mechanisms have been in- Nicotinic Acetylcholine Receptors (nAChRs) voked. Among these, a paradigmatic example is aberrant These are ligand-gated channels expressed in both neural promoter methylation of the growth regulatory genes. This and non neural tissue. They can be either homo- or hetero- mechanism is probably a common alternative to gene inacti- pentamers of a variety of and subunits that surround a vation, in human cancers. Evidence along this line is avail- pore permeable to cations, including Ca2+. nAChRs are able for channel-related genes, such as CLCA2, whose pro- potently activated by compounds present in tobacco, such as moter region is frequently inactivated by hypomethylation, nicotine and 4-(methylnitrosamino)-1-(3-pyridyl)-1-butan- in breast cancer [90]. Moreover, methylation of KCNH5 is one (NKK). Although these drugs are not directly carcino- observed in about 80% of NSCLC tissue, but only in 14% genic, current evidence suggests that nAChR activation of non-cancerous tissue [135]. Finally, inactivation of stimulates cell proliferation, angiogenesis and invasiveness CACNA1G by aberrant methylation of its 5' CpG island has [127]. Most data concern the non-small cell lung carcinoma been reported in gastric cancers, colorectal cancers and AML (NSCLC) cells, which show altered expression of nicotinic [136]. subunits in human surgical samples compared to normal Growing evidence also suggests that tumors tend to ex- tissue. Differences are also observed between smokers and press splice variants or alternative transcripts of channel- non-smokers [126]. NSCLC cells are subjected to mitogenic encoding genes, although the significance for cancer pro- effects of nicotine, apparently mediated by 7-containing gression is still uncertain. The hsloBK splice variant of gBK nAChRs [127], which are thus emerging targets for therapy. has been detected in gliomas [60] and the herg1b alternative The special involvement of 7 is suggestive, considering that 2+ transcript of Kv 11.1 is overexpressed in human leukemias this subunit confers high permeability to Ca . This interpre- and neuroblastomas [46, 137]. Another splice variant of the tation must however be taken with caution, since no func- Kv 11.1 transcript, which encodes for a C-terminus deleted tional studies exist of the properties of nAChRs in cancer Kv 11.1 protein, named herg1bUSO, is also overexpressed in cell lines [127]. Interestingly, in NSCLC cells not starved several types of tumor cell lines, and exerts a post-translation from serum, the intracellular pathways downstream to control on the membrane expression of the full length Kv nAChR activation were found to stimulate cell proliferation 11.1 protein [138]. through up-regulation of fibronectin and engagement of typical signals downstream to integrin receptors, such as the 5. CONTROL OF V AND ION FLUXES IN CANCER ERK and PI3-k/mTOR pathways [128]. Studies about the m CELLS effects of nicotine in other neoplastic cells are still in their infancy [127]. Vm in Cancer Cells Na+ Channels As has been long recognized, cycling cells are generally VGSCs are also expressed in NSCLC cells, with a possi- depolarized compared to the differentiated counterparts [139, ble role in the regulation of tumor cell invasiveness [129- 140]. This observation has resisted the advent of patch- 131]. clamp, which however considerably increased the mechanistic insight, especially when coupled with molecular biologi- 4. GENETICS AND EPIGENETICS OF CHANNEL cal techniques. The change in average Vm between dividing EXPRESSION IN NEOPLASIA and quiescent cell populations often depends on a shift in the cellular complement of ion channels. A clear example is the It is clear that most human neoplastic cell types show al- transition between cycling and differentiated glial cells, tered expression of a variety of ion channels and that these which is accompanied by a substantial hyperpolarization exert different functional roles. However, there does not because outward rectifying VGKCs are progressively substi- seem to be a specific channel-tumor correlation. This conclu- tuted by IRK channels [61]. During neoplastic transforma- 76 Current Medicinal Chemistry, 2009 Vol. 16, No. 1 Arcangeli et al. tion, Vm generally reverts to a depolarized state because ability of endothelial cells to survive and migrate in hypoxic inward rectifiers are suppressed. The extent of overlap be- and acidic environment [150] and seems to be preferentially tween the spectrum of K+ channels expressed by the neoplas- expressed on the plasma membrane of highly metastatic tic cells and those expressed during normal developmental melanoma cells [151]. The role of the H+/K+ pump in the proliferation is uncertain. For example, in some cultured acquisition of tumor-like phenotype is unclear [152]. Some neuroblastomas, the typical IRK expression of glial cells is authors believe that tumor cells have a dual benefit from substituted by that of Kv 11.1. The latter’s steady-state volt- increased proton extrusion. First, the cytoplasmic pH is age-dependent properties produce a maximal probability for maintained within proliferation-permissive values irrespec- the channel to be open around -40 mV. Therefore, a shift tive of intense glycolisis. Second, extracellular acidification from IRK- to Kv 11.1-based Vm control causes a substantial facilitates the activity of the matrix proteases that promote cell depolarization [141]. We have recently observed a simi- cell migration and invasiveness. However, this view is not lar change in human gliomas [60], which are known to unanimous and direct evidence linking abnormal proton down-regulate IRK [56]. We observe that expression of Kv pumping with the neoplastic process is lacking. It would then 11.1 has been in fact detected in early embryonic stages be premature to consider the many available proton pump [142], thus suggesting that the neoplastic condition could be, inhibitors as potential anti-cancer agents [153]. Regardless, at least in part, a reversion to a state normally occurring we notice that the functional cooperation between ion chan- during development, but the evidence is still very limited. nels, pumps and other membrane transporters in cancer cells Other VGKCs typically expressed by cancer cells, such as is still rather neglected, although it could offer interesting Kv 10.1 and Kv 1.3 (Table 1), also contribute little to the physiological insight. resting Vm at hyperpolarized values, because they activate around -30 mV. Control of Cell Volume and Motility

There is still no general explanation for these Vm changes Mammalian cells undergo complex volume changes dur- as related to the cellular state. Merely considering the aver- ing cell cycle [154,155]. These processes usually depend on age Vm is simplistic, because the expression and regulation interplay of K+ and Cl- channels and provide an explanation of many channel types, and consequently Vm, is known to of the frequent mitotic sensitivity to channel inhibitors, alter- oscillate with the cell cycle phases (for review, see [143]). native to the influences on Ca2+ fluxes. In addition, consider- Moreover, it is unclear whether (or to what extent) different ing the cooperation of ion fluxes in controlling cell motility Kv subtypes contribute to mitosis merely in virtue of their and volume, it is clear that derangement of these processes effects on Vm, or specific channels exert specific signaling may have important effects on tumor invasiveness, as seems roles. Some mechanistic hypotheses are briefly discussed in to be the case in gliomas. A full discussion of these topics is the following sections. outside our purposes and the reader is referred to the indicated reviews [12,31,61,156,157]. Interplay Between K+ Channels and Ca2+ Fluxes in the Control of Cell Proliferation 6. ION CHANNELS AND INTRACELLULAR SIG- As suggested by work in T cells, one of the K+ channel NALING roles in cancer cell proliferation could be indirect regulation 2+ of transmembrane Ca flux, through Vm modulation. Intra- Non-Conductive Roles cellular Ca2+ levels partecipate to the control of cell cycle As discussed above, some effects of ion channels on checkpoints, in normal and neoplastic proliferation [143- neoplastic progression are clearly a consequence of changes 148] and regulate other processes such as cell motility. In in ion fluxes. Less attention has been so far devoted to possi- breast cancer cells, for example, the current hypothesis is ble modulation of intracellular pathways by enzymatic roles that, in early G1, cells are depolarized and V is maintained m of channel proteins or conformational coupling with other by K 10.1 channels. Addition of GFs induces over-expre- v proteins, ultimately converging onto the transcriptional regu- ssion of K 10.1, leading to hyperpolarization and G1-S tran- v lation of cancer-related genes. These mechanisms may well sition. In turn, hyperpolarization facilitates Ca2+ influx, accompany the typical effects on ion flow. Some VGCs have which activates K 3.1 and inhibits K 10.1 via Ca2+- Ca v been in fact shown to behave as bifunctional proteins that, calmodulin. KCa 3.1 is then responsible for maintaining a besides gating ion fluxes as usual, exert an ion conduction- hyperpolarized V , which sustains the Ca2+ signal that stimu- m independent control of several intracellular responses [158- lates the cyclin-dependent kinases (CDKs) and oscillating 165]. Examples of bifunctional channels include L-type cyclins [78]. These results may explain the wide expression VGCC, whose C-terminal regions regulate transcription of Ca2+ channels in cancer cells (Table 1). [160], a TRP channel (TRP-PLIK) that contains a functional kinase domain [161], and VGSCs, whose subunits at the Role of Ion Pumps/Transporters and pH same time modulate channel function and mediate cell adhe- Early work from Serrano and co-workers showed that sion [159, 163]. An example with oncological implications is NIH3T3 fibroblasts that express a proton pump display in- the voltage sensor-containing phosphatase (Ci-VSP) of creased proliferation rate and resistance to acidic environ- Ciona intestinalis. This consists of an ion channel-like ment. Maintenance of a relatively alkaline citosol, through transmembrane domain, followed by a phosphatase domain active proton extrusion, thus appears to create a permissive highly homologous to PTEN, a tumor suppressor of human state for proliferation [149]. More recently, plasmalemmal cancers [164]. Recent review of some of these non- vacuolar (V)-ATPase function has been associated to the conducting functions is found in [165]. Targeting Ion Channels in Cancer Current Medicinal Chemistry, 2009 Vol. 16, No. 1 77

Particularly interesting evidence concerns Kv 10.1, com- flow, but on the voltage sensor conformation. For example, monly overexpressed in a variety of human cancers. In cer- cell transfection with either wild type or non-conducting tain contexts, this ion channel can be considered a true onco- mutant channels produces a similar stimulation of prolifera- gene, since transfection with Kv 10.1 tends to drive mammal- tion. These and other experiments indicate that gating of ian cells into uncontrolled proliferation and promotes tumor Kv 10.1 is directly linked to some intracellular messenger progression in cell populations injected into immuno- pathways, which include the p38 mitogen-activated protein suppressed mice [166]. Recently, it has been observed that, kinases, but not the extracellular signal-regulated kinases in murine fibroblasts and myoblasts transfected with Kv 10.1, [167]. proliferation is stimulated in a way that depends not on ion

Fig. (2). Ion channel modulation of some intracellular signaling pathways switched on by growth factors and adhesion receptors. Central box outlines the main intracellular pathways that regulate cell proliferation, survival and motility/migration. Three possibile channel roles are decsribed: signaling through modulation of intracellular Ca2+ and pH (left); signaling through interplay with growth factor receptor tyrosine kinases (middle); signaling through interaction with integrin receptors or 7 transmembrane domain (7TMD) receptors (right). Symbol legend is given in the box. 78 Current Medicinal Chemistry, 2009 Vol. 16, No. 1 Arcangeli et al.

GF Receptors and Ion Channels In childhood B-ALL, the 1/Kv 11.1 complex is triggered by adhesion onto human bone marrow stromal cells, and Cell proliferation in mammalian cells can be triggered by comprises the chemokine receptor CXCR4. In these culture GF binding to specific receptors, usually protein tyrosine conditions, B-ALL cells undergo activation of multiple sig- kinases that autophosphorylate upon ligand binding. This naling pathways, which depend on both 1 integrin and process typically turns on a kinase cascade that converges CXCR4 engagement. Blocking hERG1 currents inhibits onto phosphorylation of the extracellular signal-regulated these pathways and induces apoptotic cell death [47]. protein kinase 2 (ERK2) mitogen-activated protein (MAP) kinase. Once activated, this protein translocates to the nucleus and phosphorylates an array of specific transcription 7. ION CHANNEL PROFILE IN DIFFERENT STAGES factors [143]. A similar mechanism can also regulate ion OF NEOPLASTIC PROGRESSION channel expression and function. A few examples have been In general, K+ channel overexpression is a recurrent fea- mentioned above for the Kv channels regulated by GFs in ML-1 myeloblastic leukemia cells [35,168], as well as for ture of early cancer stages, probably because of the channel roles in cell proliferation. Later phases present more variabil- the VGSCs controlled by NGF [169] and EGF [170,171]. ity, perhaps because of the different physiological necessities But signals can also flow in the opposite direction, as sug- of neoplastic cells in different tumors. Current evidence is gested by the above-mentioned example of K 10.1, which v summarized in Fig. (3). appears to regulate some intracellular pathways typically switched on by GF receptors. Moreover, the 4-AP-sensitive Breast and prostate cancer present a similar ion channel K+ channels control ML-1 proliferation through multiple profile, in agreement with their general similarity in the signal transduction processes, such as phosphorylation of growth dependence from hormones. They are nice examples ERK 1/2 and Akt [168]. A general schema is given in Fig. of the interplay between K+ and Ca2+ channels that we have (2). illustrated [78]. The appearance of VGSCs marks instead a late stage of tumor progression, driving cell motility and Ion Channels and Cell Adhesion hence probably regulating cell invasion and metastasis. Bas- ing on work in other experimental models [163], one might Integrin-mediated cell adhesion to the extracellular ma- speculate that the VGSCs could mediate such effects by trix modulates, in proper context, cell migration, prolifera- acting as cell adhesion molecules. tion, differentiation and prevents apoptosis [172,173]. The integrin linked kinase (ILK) and the focal adhesion kinase Leukemias are characterized by early expression of (FAK) are pivotal factors in these cascades. Once activated, VGKCs, apparently necessary to permit uncontrolled prolif- it recruits further signaling components, thus leading to the eration inside the primary hematopoietic organs. A similar activation of MAP kinases, of the phosphoinositide-3 kinase pattern applies to cancers of the gastrointestinal tract, whose (PI3K), and small GTPases such as Rho A, Rac 1 and aggressive phenotype depends on their capability of growing CDC42 [173]. Fig. (2) summarizes these pathways. Adhe- and infiltrating the gastric/intestinal walls. They present a sion signals greatly overlap with those activated by GF and variety of VGKCs, such as Kv 1.3, that affect cell prolifera- cytokine receptors [174]. In some cases, such an overlap tion [112]. Once again, expression of these proliferation- depends on the formation of macromolecular complexes related channels tends to be limited to the initial steps of between integrins and the other membrane receptors, to form tumor progression, as is shown by the dramatic decrease of signaling platforms at the adhesive sites [175]. These plat- Kv 1.3 expression in advanced prostate cancers [103] and forms can also include ion channels, with ensuing crosstalk leukemias [44]. between the different components [173]. For example, T In early stages, it is possible that the K+ channel effects 2+ lymphocyte activation leads Kv 1.3 channels to associate on the [Ca ]-dependence of cell cycle is of general occur- with 1 integrins and activate them [176], an interaction also rence, although conclusive evidence is still limited to a few observed in melanoma cells [177]. A macromolecular com- experimental systems. The expression of KCa may have an plex between 1 integrin subunit and Kv 11.1 forms in both accessory role, more or less pronounced in different tumors. neuroblastoma and HEK 293 cells stably transfected with In addition, considerable evidence also points to K+ and Cl- this channel [178]. The 1/Kv 11.1 complex localizes at the channel roles in the control of cell volume during mitosis. adhesion sites, probably within caveolae/lipid rafts, and On the other hand, specific non-conductive roles on cell recruits FAK and Rac1. Both FAK phosphorylation and proliferation have yet been scarcely attended to. Rac1 activity turned out to depend on hERG1 currents [178]. Kv 11.1 is another channel widely expressed in tumors, The fact that a physical association between Kv 11.1 and 1 integrins is also observed in colorectal cancer cells1 may but its function tends to be confined to late stages of the explain why hERG1 expression is a determinant of invasive- neoplastic progression, such as transendothelial migration. In ness, in this tumor [113]. As to the overlap between signals these phases, it seems to confer protection against cytotoxic triggered by integrins and GFs, we notice that in AML cells agents, be they drugs or immune responses. Current evidence indicates that the role of Kv 11.1 in cell invasiveness is the 1/hERG1 complex also includes the VEGF receptor 1 (Flt-1). The macromolecular complex regulates signaling probably caused by its implication in the signaling cascades downstream to Flt-1 (MAP kinase and PI3K) and thus AML triggered by cell adhesion. proliferation and migration [46]. The above picture also applies to colorectal cancer, where most VGKCs are thought to control cell proliferation by modulating intracellular pH and Ca2+ signaling [109], 1Masi A et al., Unpublished data. Targeting Ion Channels in Cancer Current Medicinal Chemistry, 2009 Vol. 16, No. 1 79

Fig. (3). Expression of different ion channels during the tumor progression phases. For explanation see paragraph 5 and paragraph 7, in the main text. whereas Kv 11.1 appears to control invasiveness in later sible side effects. What is more, the recent development of stages, because of interaction with 1 integrins [173,178]. automated patch-clamp techniques has allowed the high The control of cell motility, instead, probably depends on throughput necessary for medicinal chemistry applications. complex interplay of different Kv channels, as suggested by The direct coupling of biophysical and physiopathological work on a model of epithelial colon wound healing [179]. studies in intact cells makes ion channels particularly promising targets for rational pharmaceutical screening and thera- Gliomas, lastly, are very peculiar tumors. They are highly peutic planning, although finding new candidate compounds malignant in that they rapidly infiltrate through normal tis- remains challenging [184]. sue, but lack other features of malignancy in that they do not produce metastases. Gliomas present a special ion channel Drugs can affect ion channels in different ways. They can profile that sustains their infiltration potential: They up- obstruct the conduction pore, or compete for agonist binding regulate K+ and Cl- channels in a coordinated way, with the site, or allosterically alter the probability of transition be- latter being particularly relevant to invasion and hence tween the different conformational states. Indirect mecha- probably suitable for pharmacological targeting. The peculiar nisms are also possible, because several drugs modify the profile of K+ channel expression in gliomas, which includes composition or the physical properties of the lipid bilayer, for example Kv 11.1, may reflect the necessity to exploit a which may affect channel proteins (a concise review is found full spectrum of modulatory roles, both conductive and non- in [185]). When different cell types favour different channel conductive. states, because of very different Vm dynamics, it may be possible to obtain a reasonable tissue specificity even with 8. ION CHANNELS AND THERAPY: SPECIFICITY compounds that poorly distinguish the channel subtypes AND SIDE EFFECTS [184]. This approach requires detailed understanding of drug action, which can be obtained as illustrated above. Specific Channel inhibitors would be ideal for oncologic use. examples are discussed later and the chemical structures of Many channel blockers act extracellularly, which offers the main channel inhibitors proven to affect tumor progres- several advantages: i) treatment can be calibrated more eas- sion are given in Table 3. ily; ii) metabolic effects are limited; iii) different structurally Yet, the therapeutic potential of ion channels remains related compounds can be easily tested in vitro, allowing full largely under-exploited, for two main reasons. First, the exploitation of the modern methods in cell physiology. More absence of rapid and reliable screening technologies has specifically, recent advances in the determination of 3D been a significant obstacle, until recently. This bottleneck is channel structures [180, 181] should strongly facilitate the now being bypassed by the new automated electrophysi- design of novel selective compounds [182, 183]. Once pro- ological methods. New compounds with channel-modulating duced, these can be easily tested in intact cells with patch- activity have been in fact recently applied to treat some car- clamp methods. Preliminary drug trials are usually con- diovascular, muscular, neurologic and metabolic diseases, ducted in heterologous expression systems, but tests can be such as the type II diabetes mellitus [186]. Second, except extended to more physiological conditions with relative ease. the few cases in which a channel type shows tissue-specific In this way, considerable insight can be achieved about how expression, ion channel blockers can produce serious side different compounds affect not only specific channel iso- effects. Blocking BKCa , for example, impairs smooth muscle forms and conformational states, but also complex cellular contraction, whereas inhibiting Kv 11.1 can cause fatal car- properties such as action potential firing, muscle contraction diac arrhythmias. and secretion. These studies are crucial to interpret the pos- 80 Current Medicinal Chemistry, 2009 Vol. 16, No. 1 Arcangeli et al.

230];* **[ Reference Reference *[78]; **[238] ****[239] 109]; ***[108, ****[237]

**[81]; *[33];

In vitro vitro In vivo In X X

Inhibition of cell proliferation X [78] Inhibition of cell proliferation X [109] Effect Inhibition of cell proliferation X [109] Inhibition of cell proliferation X Inhibition of cell proliferation X [240] Inhibition of cell proliferation Inhibition of cell invasion

Tumor type AML*; breast cancer** cancer** breast AML*; lines*** cell cancer colon Glioma cell lines**** Breast*; prostate** *** pancreatic lines cell endometrial cell lines****

3.4 10.1 Colon cancer cell lines Breast cancer cell lines 10.1 Colon cancer cell lines Ca v v v Channel type VGKC (un-VGKC selective blockers) channels BK Intermediate conductance K K KCNMA1 K Prostate cell lines K

3 CH O

2 N NH N

N Cl OH TRAM-34 N N N

H TEA N

F Blockers of ion channels: TEA, 4-AP, quinidine Clotrimazole; Charybdotoxin; 1-[(2- (TRAM-34) chlorophenyl)diphenylmethyl]-1H-pyrazole Iberiotoxin BDS-1 toxin Astemizole Terfenadine

Table 3. Main Inhibitors/Activators of Tumor-Associated Ion Channel and their Effect on Cancer Progression Targeting Ion Channels in Cancer Current Medicinal Chemistry, 2009 Vol. 16, No. 1 81

Reference Reference Pillozzi S., personal communication **[137] ***[241] ****[47] (Table 3). Contd…..

In vitro vitro In vivo In X *[45]

Inhibition of cell proliferation X [119] Effect Inhibition of cell proliferation and (*) invasion chemioresistance of Shortcome Inhibition of cell proliferation X

Tumor type Neuroblastoma cell lines**; Gastric lines***. cell cancer B-ALL cell lines****

11.1 Leukemia cell lines 11.1 11.1 blasts* and primary lines cell AML 11.1 Gastric cancer cell lines v v v Channel type K K K

F 3

F N H

N N N N

N O 2HCl 3 CH O O

N H Cl O

CH H N O Cl O

S O C N 3

2 H H Blockers of ion channels: E4031 Cisapride Sertindole 82 Current Medicinal Chemistry, 2009 Vol. 16, No. 1 Arcangeli et al.

242] Reference Reference [207-210] *[237] **[222]

Nu/nu Nu/nu mice X

In vitro vitro In vivo In [220] X

Effect Inhibition of cell cycle progression apoptosis of and induction Inhibition of cell invasion Phase III trial Inhibition of cell proliferation X X

Tumor type Breast and Prostate cancer cell lines Inhibition of cell invasion X [77; Glioma patients**

v 1.7) 1.7) v

currents SCLC and T leukemia cell lines v Channel type ClC-3 Glioma cell lines* VGSC Prostate cell lines K (NaVGSC Na 1.5;

O NH

2 3

CH NH HN Phenytoin O OH O OH O O O CO 3 3 H N H CH OH OH OH O H O O -phenylamides HO

C 3 HN H HN N HO CO CO 3 3 C OH H H 3 -hydroxy- H 2

-hydroxyphenylamide NH Cl

Blockers of ion channels: SP600125 inhibitor JNK TTX Phenytoin and Chlorotoxin

(Table 3). Contd….. Targeting Ion Channels in Cancer Current Medicinal Chemistry, 2009 Vol. 16, No. 1 83

is effect

or in vivo Reference Reference [213] [reviewed in in [reviewed 213] (Table 3). Contd…..

in vitro

In vitro vitro In vivo In X [229]

Effect Potentiation of cytostasis induced enterotoxins by bacterial Inhibition of cell proliferation X [211]

Inhibition of cell proliferation growth on tumor Reduction trials) and clinical mice (nu/nu Inhibition of cell proliferation X

ge SOC: Store gated TRP: channels; sodium Transient Operated Receptor Potential Channels; Potassium ffect) shows the cellular mechanisms affected by ion channel targeting drugs. Thedrugs. targeting ion by channel affected mechanisms cellular the shows ffect) The IUPHAR nomenclature is used when the channel type is defined, otherwise the common current name is is name current common the otherwise defined, is type channel the when used is nomenclature IUPHAR The ……

ssion along with their chemical structure. For toxins, no reported. or structure. For chemical is with their along sequence structure chemical ssion

olumn.

Tumor type NSCLC, glioblastoma and prostate in vitro lines cell cancer mice immunodeficient into injected Several cancer cell lines, mainly and glioblastoma NSCLC

AT-

+ +

/K /K + + Channel type Pase SOCs SOCs lines cell cancer Colon Na Na TRPM8 Melanoma cells

ATPase

O O 3 CH OH O OH H H 3 3 CH H OH

CH DIGOXIN 2 NH

H O OH 3 H O O CH 3 C O H C CH

3 H HO H B O C 3 H H

H O

O O C OH 3

HO NH H H O O

O S O

C C 3 3 HO H H HO Blockers of ion channels: (2-APB) 2-aminoethoxydiphenylborate steroids Cardiotonic (UNBS1450) 2”-oxovoruscharin of derivative hemisynthetic channels: ion of Agonists Menthol The second column (Channel type) shows the ion channel types expressed in tumor cells and tissues which are affected by drugs. drugs. by affected are which tissues and cells tumor in expressed types channel ion the shows type) (Channel column identified. second The bee has type channel ion specific no when reported, is drug the by (E affected column current ion fourth The the only observed. Sometimes been given. has drugs of effect the when tissues or cells tumor the shows type) (Tumor column third The The first column (Blockers/Agonists of ion channels) shows the main channel inhibitors/activators known to affect cancer progre cancer affect to known inhibitors/activators channel main the shows channels) ion of (Blockers/Agonists column first The reported in the two last colums. VGKC: Voltage Gated Potassium Channels; KCa: Calcium-activated Potassium Channels; VGSC: volta reported tumor cell in indicate the the third relative reference to are the References givenbrackets. Asterisks c Channels. in 84 Current Medicinal Chemistry, 2009 Vol. 16, No. 1 Arcangeli et al.

Kv 11.1, an ‘Antitarget’ rhythmics (e.g. lamotrigine and lidocaine) do not distinguish between VGSC subtypes. However, these compounds bind The case of Kv 11.1 is particularly illustrative. In cardiac preferentially to open and inactivated channels. Therefore, myocytes, it is inactivated during the depolarized plateau, treatment can be calibrated to target damaged cells that but inactivation is quickly removed on repolarization. Thus, maintain a pathologically depolarized Vm (such as the neu- inhibiting Kv 11.1 retards the cardiac repolarization, which is rons responsible for neuropathic pain), instead of cells firing reflected in prolongation of the electrocardiographic QT at high frequency. In these, the overall time spent by VGSCs interval. Uncontrolled QT interval lenghtening can result in in the open or inactive state is considerably briefer and cu- torsade de points (TdP), a life-threatening ventricular ar- mulative channel inhibition is much less effective than in rhythmia that may lead to ventricular fibrillation [187]. In steadily depolarized cells [185]. In this way, the side effects fact, many inhibitors of Kv 11.1 (hERG1 blockers), such as produced by interference with normal excitable cells are E4031, Way 123,398 and others, belong to the class III limited. A similar tactic could also work in tumors that ex- antiarrhythmic drugs. How severe is the problem is testified press VGSCs in their late stages. by the fact that reports of QT prolongation (with or without TdP), together with hepatotoxicity, determined more than Drugs addressing specific conformational states are also 60% of drug withdrawals over the last 16 years [188]. known for VGKCs [e.g. 192-194]. Kv 11.1, in particular, has voltage-dependent features that produce maximal steady Nevertheless, many lines of evidence suggest that Kv 11.1 state currents around -40 mV, a typical situation in cycling could be a useful target for oncological treatment. hERG1 cells. Hence, low doses of inhibitors binding preferentially to blockers impair neuroblastoma, leukemia and gastric cell the open channel might produce significant cumulative ef- proliferation in vitro [44, 45, 46, 119, 120, 137]. They also fects on Kv 11.1-expressing tumor cells, which present a inhibit the invasiveness of colorectal cancer cells in vitro steady fraction of open channels available for targeting with [113] and the secretion of VEGF-A in glioma [65], gastric proper drugs. Conversely, during most of the cardiac life- cancer 2, and myeloid leukemia cells [46]. Recent results time, Kv 11.1 is either deactivated (at negative Vm) or inacti- suggest that hERG1 blockers produce similar effects in vivo. vated (during the depolarized plateau) and drugs blocking Treating nude mice with E4031 for two weeks, after subcu- the pore should be less harmful for cardiac function. A de- taneous injection of human gastric cancer cells, significantly tailed study about the frequency-dependency of hERG1 decreased tumor growth and the intratumoral total vascular 2 blockade for different compounds has been recently carried area . Similar results were obtained in a leukemic model in out by Stork and colleagues [193]. As reminded above, how- vivo. Myeloid or lymphoblastic leukaemia cells were in- ever, a supplementary problem presented by these drugs is jected into NOD-SCID mice that were subsequently treated that they usually bind to the intracellular side of the channel for two weeks with E4031. Treatment decreased bone mar- protein. To develop drugs that bind to the extracellular side row (BM) engraftment and peripheral blood (PB) invasion, 2 and present conformational specificity, efforts should proba- with ensuing increase in mice survival . Therefore, hERG1 bly be directed towards the array of peptide toxins specific blockers seem to be well worth considering for treatment of for Kv 11.1. The first was discovered in 1999 and new types some neoplasias. have been found almost yearly. All of these bind extracellu- However, most hERG1 blockers present the worst possi- larly with excellent specificity and bind different channel ble combination of drawbacks: they bind the channel in- portions. The scorpion toxins BeKm-1 and CnErg1 (ErgTx1) tracellularly and produce grave side effects. Many drugs occupy the external vestibule in the closed state. They are inhibit Kv 11.1 (reviewed in [189]). Aside from class III gating modifiers in that they shift the channel activation anthyarrhythmics, we recall some antihistaminics (e.g. ter- curve in the positive direction. Moreover, they obstruct, to fenadine), prokinetics (e.g. cisapride), antipsychotics (e.g. different extent, the pore conductance when the cannel is sertindole) and antibiotics (e.g. erythromycin) [190]. The open [195, 196]. Their binding site is not identical, with molecular reasons for this ‘promiscuousness’ reside in the BeKm-1 probably targeting a deeper pore region. BeKm-1 structural features of the inner channel cavity, to which most and CnErg1 do not seem to distinguish the different hERG of these drugs bind. For details we refer the reader to recent isoforms, whereas the sea anemone toxin APETx1 is specific reviews [188, 191]. The following paragraphs describe po- for Kv 11.1 [197]. APETx1 is also a gating modifier, but tential approaches to circumvent these and other problems binds the S3b helix, instead of the channel vestibule [198]. A arising when targeting ion channels in oncologic therapy. summary of the compounds known to inhibit Kv 11.1 is given in Fig. (4). Hence, further studies could exploit these Targeting Different Conformational States or Different observations to produce compounds more selective for the Channel Regions different isoforms or conformational states.

Because neoplastic cells are characterized by Vm dynam- Finding Molecules with Higher Specificity ics totally different from that observed in excitable cells, the proportion of time spent by a VGC in a given state can be This approach is useful when the side effects depend on very different in normal and cancer cells. In principle, a drug cross-reaction of available drugs. Among recent successes, that preferentially binds one of these conformational states we mention the results of K.G. Chandy and coworkers, who could select among different cell types. Such a therapeutic selectively targeted the Kv 1.3 of effector/memory T (TEM) strategy has been successfully applied, in a different physio- cells, which is implicated in inflammatory autoimmune dis- logical context, to VGSCs. Some anticonvulsants and antiar- eases such as multiple sclerosis and rheumatoid arthritis [199-202]. A long-term loss of TEM cells, with consequent 2Lastraioli, E.; Pillozzi, S. Unpublished data. improvement of autoimmune disease symptoms, was ob- Targeting Ion Channels in Cancer Current Medicinal Chemistry, 2009 Vol. 16, No. 1 85

Fig. (4). Summary of different inhibitors of Kv 11.1 (hERG1) channels and their mode of action. Figure lists some of the specific toxins isolated to date, a bloking monoclonal antibody [47] and (on the right) the main classes of non peptidic blockers. Box illustrates the structure of a Kv 11.1 subunit, highlighting some of the sensitive residues for channel block. For description of toxin action, see the main text. tained by treating mice with a modified form of the sea minimal acute toxicity. Several novel -hydroxy-- anemone toxin ShK (ShK-186), with higher selectivity for phenylamides, designed by computational methods, were Kv 1.3. Importantly, ShK-186 spares the T-cell population tested in vitro. Compared to phenytoin, they presented higher that confers protection against infection and cancers [203]. inhibitory effects on both Na+ currents and prostate cancer Care must be taken, however, because TEM cells are protec- cell growth [207, 208]. These derivatives are currently being tive in some cancers [204,205] but not others [206]. Hence, tested for their antitumor activity in human prostate cancer the chronic blockade of Kv 1.3 could worsen the prognosis of xenografts [209]. some tumors. Seeking highly specific compounds may be helpful also These and other promising results suggest that similar in the case of Kv 11.1. The level of torsadogenic potential of approaches could be applied to oncologic treatment. The channel blockers is often unknown, because the mechanistic simplest rationale follows from the observations in vitro relationship between QT prolongation and occurrence of suggesting that ion channel blockers can inhibit cell prolif- arrhythmias is poorly understood. Some drugs, such as the eration [9]. Now that individual channel types can be as- antipsychotic sertindole, inhibit Kv 11.1 (and prolong QT) signed to specific cancer types, at least in some cases, the without producing torsadogenic effects and are thus adminis- race is open to search for more specific blockers. tered safely [189]. Because sertindole has particularly high affinity for K 11.1 [210], it is possible that the torsadogenic Treatment of metastatic prostate cancer has been at- v effect of many hERG blockers depends on their lower speci- tempted by searching new VGSC blockers. A rational drug design based on the phenytoin binding site has been adopted ficity, which may produce cross-reactions with other ion channels that cooperate in producing torsadogenicity. In this to develop novel inhibitors with enhanced activity and 86 Current Medicinal Chemistry, 2009 Vol. 16, No. 1 Arcangeli et al. case, the search of more specific compounds, basing on the neous classes of inhibitors usually lack subtype specificity. structure of sertindole (see Table 3), seems well worth at- For instance, many compounds are known to block Kv 10.1, tempting. but none of them is specific. However, the monoclonal antibodies against K 10.1 produce specific inhibition in intact Finally, the TRPM8-encoding gene is up-regulated in v prostate cancer and other malignancies (see above). Classical cells and partial reduction of tumor growth in vivo, in immunodeficient mice in which epithelial tumor cells were in- agonists of TRPM8 have been reported to suppress prolifera- jected subcutaneously [214]. These results indicate that spe- tion of melanoma cells [211] and their effects have been cific inhibition of some channel types could have therapeutic recently characterized [212]. value. Other potentially useful blocking antibodies have been The general points made for ion channels also apply to raised against epitopes located in the external vestibule of targeting ion pumps. A representative example is offered by Kv 1.2 and Kv 3.1 [217]. More recently, a rabbit anti-rat + + the work performed on the Na /K ATPase. The pump’s 1 TRPV1 polyclonal antibody was found to partially antago- subunit is overexpressed in some cancers, especially NSCLC nise the channel functions [218]. and glioblastomas (Table 1, reviewed in [213]). Cardiotonic A common method to generate blocking antibodies is ad- steroids (cardenolides) are natural ligands and inhibitors of + ressing epitopes on the third extracellular region (E3) of an the Na pump but they lack sufficient anti-tumor activity to be used as single anti-cancer agents at clinically acceptable ion channel. E3-targeted anti TRPC5 antibodies, for instance, are very useful extracellular inhibitors of this chan- doses. However, a hemisynthetic derivative of 2”-oxovo- nel, for which good blockers are unknown [219]. E3 target- ruscharin (UNBS1450) displays unique structural features ing was also applied to VGSCs, leading to a subtype-specific that make its binding to 10 to 100 times stronger than that 1 inhibitor of Na 1.5 [220]. Finally, antibodies raised against of the classical cardenolides. UNBS1450 leads to cell death v + surface proteins may turn out to modulate, unexpectedly, in gliomas that overexpress the Na pump and dramatically impairs NSCLC cell growth in vitro. Its effects in vivo are some ion channels. An example is rituximab, a monoclonal antibody for CD20 currently used to treat B cell large cell presently under study in preclinical trials [213]. 2+ lymphomas. It was found to significantly decrease [Ca ]i and to inhibit K 3.1 currents, with consequent inhibition of Delivering Traceable or Cytotoxic Compounds Ca cell proliferation [37]. Another interesting example is When the side effects depend on drug interference with SP600125, which is not an antibody. This compound notori- the channel of interest in non tumoral tissue, finding more ously blocks the JNK kinase, but has also been reported to specific or more efficacious compounds may produce little inhibit the Kv 1.5 channels, in a JNK-independent way [220]. benefit. Channel inhibition by specific drugs is also of little avail when the effects on malignancy are only partial. None- Alternative Molecular Tools: Antisense Oligonucleotides theless, even in these cases, the development of specific (AO) and siRNAs drugs offers important advantages. First, these compounds This novel and promising approach could be applied to can be made traceable, e.g. by incorporating radiohalogen target ion channels, so as to bypass the adverse effects of non atoms, with obvious implications for diagnostic use. Second, specific channel expression in neoplastic tissue. First, highly they may be used to specifically deliver cytotoxic molecules. proliferating neoplastic cells are more liable to be transfected K 10.1 is a well studied example of both possibilities. It v with exogenous DNAs/RNAs, compared to quiescent nerv- presents significant expression outside the central nervous ous and cardiac cells. Second, mRNA targeting might be system only during the progression of particular tumors. more effective in addressing cycling cells, whose membrane Effective therapy is thus possible if one can produce drugs protein complement is rapidly turning over. An application that i) do not cross readily the blood-brain barrier and ii) are - of antisense technology is offered by Cl channels. As previ- sufficiently specific not to present significant cross-binding ously described, CLIC4 expression increases in keratinocytes with other channel proteins. As further discussed in the next exposed to DNA-damaging agents. In a squamous cancer section, monoclonal antibodies have been generated against cell line, when the CLIC4 level is reduced by expressing an K 10.1, by Pardo and colleagues. Apart from anti-tumor v inducible CLIC4 antisense oligonucleotide, cells undergo efficacy, which seems limited, these antibodies can be very apoptosis. In tumors derived from transplanting these cells useful for both diagnosis and delivering of cytotoxic drugs into nude mice, application of the CLIC4 antisense oligonu- [214]. Alternatively, monoclonal antibodies against cancer- cleotide inhibits tumor growth, increases apoptosis and re- specific surface proteins, can bring specific channel blockers duces proliferation. This effect is potentiated by intraperito- directly on neoplastic tissue, in order to spare nearby normal neal co-administration of TNF [221]. cells or distant tissues. This approach has been developed and is currently used to drive cytotoxic drugs and toxins Use of Modified Toxins directly to cancer cells [215, 216]. This method clearly indicates the necessity of ever more detailed profiling of cancer As briefly discussed for hERG1, peptide toxins are specific ion channels, and membrane proteins in general. known for many ion channels or channel-associated proteins. Toxins are often very selective. They can be modified for Alternative Molecular Tools: Blocking Antibodies specific purposes and used for inhibiting specific channels or delivering cytotoxic compounds, or both. For instance, in VGKC and TRP channels are often formed by different human gliomas, ClC-3 channels are implicated in cancer subunits, encoded by different genes. This explains the great spread. They can be inhibited indirectly, but specifically, by variety of channel and current features. In general, homoge- the scorpion toxin chlorotoxin (Cltx). Cltx binds a membrane Targeting Ion Channels in Cancer Current Medicinal Chemistry, 2009 Vol. 16, No. 1 87 bound metalloproteinase that moves within the membrane REFERENCES and produces inhibition of ClC-3 [62]. A recently developed radiolabeled Cltx compound (I131-Cltx) has successfully [1] De Vita, V.; Hellmann S.; Rosenberg, S. Cancer. 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Received: September 09, 2008 Revised: November 15, 2008 Accepted: November 18, 2008