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1-O-hexadecyl-2-O-methyl-rac-glycero-3-β-lactose; PAF, platelet-activating factor; PC, phosphocholine; Perifosine, (1,1-dimethyl-piperidin-1-ium-4yl) octadecyl ...
JPT-06915; No of Pages 18 Pharmacology & Therapeutics xxx (2016) xxx–xxx

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Pharmacology & Therapeutics journal homepage: www.elsevier.com/locate/pharmthera

Associate editor: M. Birrell

Alkyl ether lipids, ion channels and lipid raft reorganization in cancer therapy Paul-Alain Jaffrès a,e, Consuelo Gajate b, Ana Maria Bouchet c,e, Hélène Couthon-Gourvès a,e, Aurélie Chantôme c,e, Marie Potier-Cartereau c,e, Pierre Besson c,e, Philippe Bougnoux c,d,e, Faustino Mollinedo b, Christophe Vandier c,e,⁎ a

CEMCA, UMR CNRS 6521, IBSAM, Université de Brest, 6, Avenue V. Le Gorgeu, 29238 Brest Cedex 3, France Laboratory of Cell Death and Cancer Therapy, Department of Cellular and Molecular Medicine, Centro de Investigaciones Biológicas, Consejo Superior de Investigaciones Científicas (CSIC), C/Ramiro de Maeztu 9, E-28040 Madrid, Spain c INSERM/University of Tours U1069, Nutrition-Croissance et Cancer (N2C), F-37032 Tours, France d Centre HS Kaplan, CHU Bretonneau, Tours F-37032, France e Network “Ion channels and Cancer-Canceropole Grand Ouest”, (IC-CGO), France b

a r t i c l e

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a b s t r a c t Synthetic alkyl lipids, such as the ether lipids edelfosine (1-O-octadecyl-2-O-methyl-rac-glycero-3phosphocholine) and ohmline (1-O-hexadecyl-2-O-methyl-rac-glycero-3-β-lactose), are forming a class of antitumor agents that target cell membranes to induce apoptosis and to decrease cell migration/invasion, leading to the inhibition of tumor and metastasis development. In this review, we present the structure–activity relationship of edelfosine and ohmline, and we point out differences and similarities between these two amphiphilic compounds. We also discuss the mechanisms of action of these synthetic alkyl ether lipids (involving, among other structures and molecules, membrane domains, Fas/CD95 death receptor signaling, and ion channels), and highlight a key role for lipid rafts in the underlying process. The reorganization of lipid raft membrane domains induced by these alkyl lipids affects the function of death receptors and ion channels, thus leading to apoptosis and/or inhibition of cancer cell migration. The possible therapeutic use of these alkyl lipids and the clinical perspectives for these lipids in prevention or/and treatment of tumor development and metastasis are also discussed. © 2016 Elsevier Inc. All rights reserved.

Keywords: Alkyl ether lipids Lipid rafts Ion channel Death receptor Apoptosis Cell migration

Contents 1. 2. 3. 4. 5.

History . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Structure–activity relationship of edelfosine and ohmline and their effects on the SK3 channel Mechanism of action of edelfosine and ohmline . . . . . . . . . . . . . . . . . . . . . Importance of lipid rafts for edelfosine and ohmline's antitumor effect . . . . . . . . . . . Clinical therapeutic use of edelfosine, alkylphosphocholines, and ohmline in cancer . . . . .

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Abbreviations: AEL, alkyl ether lipid; Bid, BH3-interacting domain death agonist; CASMER, cluster of apoptotic signaling molecule-enriched rafts; CCT, CTP:phosphocholine cytidylyiltransferase; DISC, death-inducing signaling complex; DMSO, dimethyl sulfoxide; Edelfosine, 1-O-octadecyl-2-O-methyl-rac-glycero-3-phosphocholine or ET-18-OCH3; FADD, Fas-associated death domain-containing protein; HPLC/MS, high pressure liquid chromatograph/mass spectrometry; IKCa, intermediate conductance calcium-activated potassium channels; JNK, c-Jun amino-terminal kinase; KCa, calcium-activated potassium channels; Kir2.2, isoform 2.2 of potassium inward rectifier; LPA, lysophosphatidic acid; LPC, lysophosphatidylcholine; Miltefosine, hexadecylphosphocholine; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide; NSCL, non-small cell lung carcinoma; Ohmline, 1-O-hexadecyl-2-O-methyl-rac-glycero-3-β-lactose; PAF, platelet-activating factor; PC, phosphocholine; Perifosine, (1,1-dimethyl-piperidin-1-ium-4yl) octadecyl phosphate; PLC, phospholipase C; PKCs, protein kinase C (different isoforms); RANKL, receptor activator of nuclear factor kappa-B ligand; SKCa, small conductance calcium-activated potassium channels; STIM, stromal interaction molecule; TRAAK, twik-related arachidonic acid-stimulated potassium channels; TREK-1, twik-related potassium channel-1; TRPC, transient receptor transient channels; TWIK, tandem pore domain weak inward rectifier potassium channels. ⁎ Corresponding author at: INSERM/University of Tours U1069, Nutrition-Croissance et Cancer (N2C), F-37032 Tours, France. Tel.: +33 247366024; fax: +33 247366226. E-mail address: [email protected] (C. Vandier). URL's: http://www.ic-cgo.fr (P.-A. Jaffrès), http://www.ic-cgo.fr (A.M. Bouchet), http://www.ic-cgo.fr (H. Couthon-Gourvès), http://www.ic-cgo.fr (A. Chantôme), http://www.ic-cgo.fr (M. Potier-Cartereau), http://www.ic-cgo.fr (P. Besson), http://www.ic-cgo.fr (P. Bougnoux), http://www.ic-cgo.fr (C. Vandier).

http://dx.doi.org/10.1016/j.pharmthera.2016.06.003 0163-7258/© 2016 Elsevier Inc. All rights reserved.

Please cite this article as: Jaffrès, P.-A., et al., Alkyl ether lipids, ion channels and lipid raft reorganization in cancer therapy, Pharmacology & Therapeutics (2016), http://dx.doi.org/10.1016/j.pharmthera.2016.06.003

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6. Administration route and formulations for the putative clinical use of edelfosine and ohmline 7. Clinical perspectives . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8. Conclusion and perspectives . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Conflict of interest statement . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Acknowledgments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

1. History In the early 1960s, it was found that the generation of 2lysophosphatidylcholine (LPC) by phospholipase A2 and its accumulation in the macrophage membrane led to activation and enhancement of the phagocytic activity of peritoneal macrophages in vitro and in vivo (Munder et al., 1969; Munder & Modolell, 1973). Thus, LPC was suggested to play a role in the defense mechanism of the immune system through macrophage activation, but LPC was not stable, and was biologically inactivated either by the action of acyltransferase, leading to phosphatidylcholine, or by lysophospholipase, leading to glycerophosphocholine (Mulder and van Deenen, 1965). To circumvent these metabolic changes, LPC analogues with longer half-life times were synthesized as a result of a joint effort of different groups led by Herbert Fisher, Otto Westphal, Hans Ulrich Weltzien and Paul Gerhard Munder in the Max-Planck-Institut für Immunbiologie in Freiburg. A major emphasis was laid on modifications in the positions C1 and C2 of the glycerol backbone in the molecule of LPC (1-acyl-sn-glycero-3phosphocholine), replacing the ester bonds for ether linkages to generate alkyl ether lipids (AELs) that were unable to be metabolized by either acyltransferases or lysophospholipases. As a result, edelfosine (1-O-octadecyl-2-O-methyl-rac-glycero-3-phosphocholine, ET-18OCH3), was synthesized by Günter Kny in 1969, making use of the previous experience of Bernd Arnold and Hans Ulrich Weltzien with the synthesis of 1-O-alkyl and 2-O-methyl derivatives of glycerol. As expected, a number of the newly synthesized ether analogues of LPC were potent immune modulators (Munder et al., 1979), and interestingly enough some of them turned out to exert strong antitumor activity (Tarnowski et al., 1978; Munder, 1982), with edelfosine being the most active of these compounds. Subsequent work conducted in the late 1970s and 1980s by Munder's team and additional groups showed that the antitumor action of edelfosine was due to both an enhanced tumoricidal activity of macrophages and to a direct cytostatic and cytotoxic effect on tumor cells (Munder et al., 1979; Andreesen et al., 1984; Scholar, 1986). Later in 1993, edelfosine was shown independently by researchers in Madrid (Spain) (Mollinedo et al., 1993) and Milan (Italy) (Diomede et al., 1993) to promote apoptosis in cancer cells. Then, in the late 1990s and early 2000s, a number of findings were unveiled by Faustino Mollinedo and Consuelo Gajate's group in Valladolid and Salamanca (Spain) showing edelfosine-induced selective apoptosis in cancer cells, following the preferential drug uptake in tumor cells (Mollinedo et al., 1997; Gajate et al., 2000a, 2000b) as well as the reorganization of membrane raft domains (Gajate & Mollinedo, 2001; Gajate et al., 2004). Thus, these data provided the first evidence for a selective proapoptotic drug and for the involvement of membrane rafts in cancer chemotherapy. A number of compounds derived from the original AELs have been synthesized, including miltefosine (hexadecylphosphocholine). It is a simplified version of the above AELs and lacks the glycerol backbone (Unger et al., 1988; Eibl & Unger, 1990; Unger et al., 1990). Miltefosine is used in the clinic as a topical treatment (Miltex; Asta Medica, Frankfurt, Germany) for cutaneous metastases in breast carcinoma (Clive et al., 1999; Smorenburg et al., 2000; Leonard et al., 2001). Replacement of the choline head group in miltefosine by a cyclic aliphatic piperidyl moiety yielded another set of compounds with an improved therapeutic index, from which perifosine (1,1-dimethyl-piperidin-1-ium-4-yl) octadecyl phosphate) stood out for its potent and promising

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antitumor activity against various cancer cell types and it is being currently tested in clinical trials (Richardson et al., 2012). These new compounds are also known as alkylphosphocholine analogues, which together with additional structurally related compounds constitute a family of synthetic compounds collectively named as alkylphospholipid analogues, and include rather heterogeneous chemical structures, including AELs, but edelfosine is still considered as the prototype of this increasing family of alkylphospholipid compounds. Since the late 1990s additional promising compounds have introduced carbohydrates or carbohydrate-related molecules to the AEL chemical leads, leading to the so-called glycosylated phospholipids (Danker et al., 2010; Semini et al., 2014) and the non-phosphorus glycosylated ether lipids (Arthur & Bittman, 2014). Beyond the action of edelfosine and glycosylated phospholipids as proapoptotic drugs, a new mode of action was recently discovered with the observation that, at lower concentration, edelfosine interacted with the SK3 channel (Potier et al., 2011) leading to a reduction of SK3-dependent cancer cell migration. A less toxic analogue of edelfosine was identified, ohmline (1-O-hexadecyl-2-O-methyl-racglycero-3-β-lactose (Girault et al., 2011; Chantome et al., 2013) which is a glyco–glycero ether lipid. Because of its apparent lack of general toxicity, its in vivo use revealed its ability to prevent bone metastases in a metastatic breast cancer model (murine experiments). The anticancer action of edelfosine (proapoptotic effect) or ohmline (anti-metastatic effect) is based on different mechanisms of action but, to some extent, they also possess some similarities since they interact selectively with proteins localized at the plasma membrane and more likely in the lipid rafts. The recent insights in the mechanisms of action of both edelfosine and ohmline are further detailed and discussed below, including the potential medical innovations suggested by these original modes of action. 2. Structure–activity relationship of edelfosine and ohmline and their effects on the SK3 channel The mode of action of edelfosine and ohmline is singular compared to most anticancer drugs. These lipids do not interact with DNA. Because of their amphiphilic nature, they incorporate into cell membranes where they can affect a large number of membrane-embedded proteins. Among them, ion channels and more precisely the SK3 potassium channel was found to be sensitive to AELs (Girault et al., 2011). The SK3 channel belongs to the calcium-activated potassium channel (KCa) family that comprises many channels which differ in their primary amino acid sequences and exhibit different single channel conductance (Wei et al., 2005). KCa channels can be divided into three subfamilies: big conductance (BKCa), intermediate conductance (IKCa) and small conductance (SKCa). IKCa is also named as KCa3.1 or SK4 or IK1. SK3 is a member of the SKCa ion channel family that includes 2 other isoforms SK1 and SK2. The SK3 channel (like SK1, SK2 and IKCa channels), through its high calcium sensitivity (it is activated by submicromolar concentrations of intracellular calcium), plays a role in the regulation of signaling pathways involving calcium, in both excitable and nonexcitable cells. In 2010s, Paul-Alain Jaffrès's group in Brest (France) and Christophe Vandier's group in Tours (France) demonstrated the capacity of AELs to reduce cancer cell migration and metastasis development by targeting the SK3 channel leading these groups to propose AELs and derivatives as a new class of anti-metastatic drugs in targeted and personalized cancer therapy (Girault et al., 2011; Chantome et al., 2013). Therefore, the modulation of SK3 channel activity constitutes a

Please cite this article as: Jaffrès, P.-A., et al., Alkyl ether lipids, ion channels and lipid raft reorganization in cancer therapy, Pharmacology & Therapeutics (2016), http://dx.doi.org/10.1016/j.pharmthera.2016.06.003

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Fig. 1. Structure of edelfosine and its analogues featuring a phosphocholine polar head.

new opportunity in the context of cancer therapy. The inhibition of SK3 channel activity can be achieved by some peptides (e.g. apamin) or heterocyclic-based compounds (e.g. (R)-N-(benzimidazol-2-yl)1,2,3,4-tetrahydro-1-naphtylamine, NS8593) but these compounds present side effects (neurotoxicity for apamin or a low selectivity of action for many heterocyclic-based compounds) (Girault et al., 2012). The evaluation of amphiphilic compounds, such as edelfosine, as potential modulators of the SK3 channel was recently assessed (Girault et al., 2011; Potier et al., 2011). In vitro and in vivo evidence have shown that edelfosine induces apoptosis in a variety of tumor cells when used at the 6–10 μM range (Mollinedo et al., 1997; Gajate et al., 2000a; Gajate & Mollinedo, 2007), which corresponds to the concentration reached in plasma in animal model studies (Estella-Hermoso de Mendoza et al., 2009; Mollinedo et al., 2010a, 2010b). Edelfosine can modulate the activity of the SK3 channel in an acute manner at 10 μM (measured by the patch clamp technique to monitor the modulation of SK3 channel activity) leading to at least 50% reduction in SK3 current (Girault et al., 2011). Edelfosine-C18 applied at 300 nM after 24 h (chronic effect) reduced cancer cell migration by 60% after 24 h treatment (Fig. 1, Table 1). It is worth noticing that the reduction by two carbon atoms of the alkyl chain of edelfosine (edelfosine-C16 or ET-16OCH3 or 1-O-hexadecyl-2-O-methyl-rac-glycero-3-phosphocholine) produced the same effect on cancer cell migration (Girault et al., 2011), an effect that was not due to their cytotoxic effect. The main difference in terms of biological effects, when edelfosine-C16 and edelfosine-C18 were compared, was their cytotoxicity that was evaluated on MDA-MB-435s cancer cell line and non-cancer MCF-10A and 184A1 cell lines. Both compounds present some cytotoxicity at 10 μM (evaluated by MTT tests), but edelfosine-C18 was more cytotoxic than edelfosine-C16. Other compounds possessing a chemical structure closely related to edelfosine were assessed for their action on the SK3 channel. Platelet-activating factor (PAF, 1-O-hexadecyl-2-O-acetyl-racglycero-3-phosphocholine, compound 2, Fig. 1), which differs from edelfosine by the replacement of the methoxy group at the sn2 position of the glycerol moiety by an acetyl group (Fig. 1), had an activating effect on the SK3 channel in the acute test and no effect on SK3dependent cell migration (Fig. 1, Table 1). Further investigations on

the effect of the substitution at the sn2 position of the glycerol moiety led to evaluation of compound 3 (1-O-hexadecyl-rac-glycero-3phosphocholine or LysoPAF). This compound which was used only for the chronic test was inactive. Miltefosine (hexadecylphosphocholine; compound 4), which is also an anticancer drug (Danhauser-Riedl et al., 1990; Hilgard, 1990; Unger & Eibl, 1991), is formed by an alkyl chain (C18) directly bonded to the phosphocholine polar head without glycerol moiety. Miltefosine (Fig. 1, Table 1, compound 4) reduced SK3dependent cell migration (−20% at 300 nM), but at a significantly lower extent than edelfosine. The addition of a methoxy group on the alkyl chain at a position close to the polar head (Fig. 1, 2-methoxyhexadecylphosphocholine, compound 5) greatly increased the capacity to reduce SK3-dependent cell migration and no toxicity was observed at 10 μM. Finally, the role of the polar head appeared essential since compounds 6 (1-O-hexadecyl-2-O-methyl-rac-glycerol) and 7 (1-Ohexadecyl-rac-glycerol) were ineffective in modulating SK3-dependent cell migration (Fig. 1, Table 1).

Table 1 Effect of edelfosine and its analogues on SK3 current and SK3-dependent cancer cell migration. Data are from Girault et al. (2011).

1 (edelfosine C18) 2 (PAF) 3 (lysopaf) 4 (miltefosine) 5 6 7

% of SK3 current modulation at 10 μMa

% of inhibition cell migration at 300 nMb

−50% +30% ND ND ND ND ND

60% No effect No effect 20% 50% No effect No effect

a Acute test: current recorded at 0 V in HEK-293 cells expressing the recombinant SK3 channel (ratio of the current in the presence of tested compound to the current without the presence of compound). b Chronic test: reduction of MDA-MB-435s cell migration at 300 nM for 24 h (% determined as the ratio of the number of cells that have migrated in the presence of tested compound to the number of cells that have migrated without the compound). The SK3dependent part of MDA-MB-435s cell migration is 60%. ND: not determined.

Please cite this article as: Jaffrès, P.-A., et al., Alkyl ether lipids, ion channels and lipid raft reorganization in cancer therapy, Pharmacology & Therapeutics (2016), http://dx.doi.org/10.1016/j.pharmthera.2016.06.003

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The toxicity of the most efficient modulator of the SK3 channel presented in Fig. 1 (edelfosine 1 and compound 5) led to the syntheses of new structures that would exhibit a lower toxicity. It is worth noticing that compounds 6 and 7, which were devoid of the phosphocholine polar head, were inefficient to reduce SK3-dependent cell migration but, unlike edelfosine and compound 5, they were not toxic at 10 μM (Girault et al., 2011). This result suggested that the toxicity could be due to the phosphocholine polar head. However, phosphocholine (PC) moiety is a very common polar group present in the structure of a large panel of natural phospholipids, thus suggesting that this polar head group cannot be intrinsically associated to a systematic toxicity. The toxicity could arise from the association of PC polar head, which is a highly polar head group (zwitterionic moiety), with a hydrophobic domain featuring the presence of only one lipid chain. This association generated a molecule that has marked detergent properties. This property likely explains the hemolytic side effect of edelfosine (Ahmad et al., 1997). These features encouraged to develop amphiphilic compounds with a hydrophilic moiety possessing a less pronounced polar character. The second point of concern to propose new analogues of edelfosine was based on the observation that oat can replace, to some extent, phospho–glycero–lipids by glyco–glycero–lipids (mono- or polysaccharide-based lipids) when the plant grows with a deprivation in phosphorus (Andersson et al., 2005). This observation suggests that the saccharide-based glycero–lipid could mimic phosphocholine-

based-glycero–lipids. This second observation is also consistent with the first point of consideration since a monosaccharide or polysaccharide unit is indeed less polar than a zwitterionic polar head group of edelfosine. On these bases and thanks to the impulse of Dr. G. Simon, Dr. J.P. Haelters and Pr. B. Corbel from the University of Brest, new series of compounds were synthesized in which the PC polar head group was replaced by a monosaccharide or disaccharide unit. Fig. 2 shows the first analogues of edelfosine having a monosaccharide (glucose, galactose) or a disaccharide unit. These compounds were prepared by a glycosylation step between the activated and protected saccharide unit and a lipid-based alcohol (Girault et al., 2011). The first surprise was the observation that compound 8Ac (1-O-hexadecyl-2-O-methyl-rac-glycero3-β-glucosetetraacetate) which possessed a glucose moiety protected with four acetyl groups, was almost as efficient as compound 8 (1-Ohexadecyl-2-O-methyl-rac-glycero-3-β-glucose; the compound that features four free alcohol functional groups on the glucose unit) in reducing SK3-dependent cell migration (Fig. 2, Table 2). The inhibition of cell migration was modest (respectively −25% and −20%), but this result indicated that both protected- (acetyl groups) and deprotectedbased amphiphiles were worthy of being tested. The replacement of the glucosyl moiety with a galactosyl unit (1-O-hexadecyl-2-O-methyl-rac-glycero-3-β-galactosetetraacetate 9Ac and 1-O-hexadecyl-2-Omethyl-rac-glycero-3-β-galactose 9) offered almost the same results (20% reduction of cell migration at 300 nM after 24 h). The replacement

Fig. 2. Structure of glyco–ether lipid.

Please cite this article as: Jaffrès, P.-A., et al., Alkyl ether lipids, ion channels and lipid raft reorganization in cancer therapy, Pharmacology & Therapeutics (2016), http://dx.doi.org/10.1016/j.pharmthera.2016.06.003

P.-A. Jaffrès et al. / Pharmacology & Therapeutics xxx (2016) xxx–xxx Table 2 Selection of some glycerol–ether lipids that were synthesized and then tested on SK3 current and SK3-dependent cancer cell migration. Data are from Girault et al. (2011).

8Ac 8 9Ac 9 10Ac 10 (ohmline) 11 (ohmline-C18) 12Ac 12

% of SK3 current modulation at 10 μMa

% of inhibition cell migration at 300 nMb

ND ND −48% ND ND −73% ND ND ND

25% 20% 20% 20% 30% 50% 30% 44% 44%

ND: not determined. a Acute test: current recorded at 0 mV in HEK-293 cells expressing the recombinant SK3 channel (ratio of the current in the presence of tested compound to the current without the compound). b Chronic test: reduction of MDA-MB-435s cell migration at 300 nM for 24 h (% determined as the ratio of the number of cells that have migrated in the presence of tested compound to the number of cells that have migrated without the compound). The SK3dependent part of MDA-MB-435s cell migration is 60%.

of the mono-saccharide moiety by a disaccharide unit generated new effective edelfosine analogues. In particular, compound 10 (1-Ohexadecyl-2-O-methyl-sn-glycero-3-β-lactose or ohmline), that features a lactosyl unit, reduced SK3 current by 73% and SK3-dependent cell migration by 50%. The equivalent molecule possessing protected hydroxyl groups of the lactosyl unit with the acetyl groups (compound 10Ac) was less efficient as revealed by the SK3-dependent cell migration assays (−30% of cell migration instead of −50% for ohmline 10; Table 2). Of note, the lengthening of the lipid chain of compound ohmline by two carbon atoms to define compound 11 (1-O-octadecyl2-O-methyl-rac-glycero-3-β-lactose) was also less efficient than ohmline (Fig. 2, Table 2). Finally, the replacement of the (1 → 4) connected disaccharide unit (lactose) present in ohmline by a (1 → 6)based disaccharide (melibiose) also produced efficient compounds (1-O-hexadecyl-2-O-methyl-rac-glycero-3-β-melibiose-heptaacetate 12Ac and 1-O-hexadecyl-2-O-methyl-rac-glycero-3-β-melibiose 12) and exhibited similar activities when the hydroxyl groups are protected or not with the acetyl groups (reduction of cell migration by 44%). Recently, we assessed the effect of the stereochemistry of the anomeric position of the lactose unit bonded to the glycerol moiety. For ohmline, the lactose unit is bonded to the glycerol unit with a β configuration. By using trimethylsilane as protecting group we prepared α-ohmline (1-Ohexadecyl-2-O-methyl-sn-glycero-3-α-lactose; Fig. 2 compound 10bis) (Berthe et al., 2016). We found that α-ohmline was not able to modulate the SK3 channel at 10 μM (acute test involving patch clamp measurement). The toxicity for the compounds displayed in Fig. 2 was assessed by MTT tests (Girault et al., 2011). Compounds 9Ac, 9 (galactose-based amphiphiles), ohmline-C18 11 and 12Ac (heptaacetylated melibiose derivative) were toxic at 10 μM whereas compounds 8Ac and 8 (glucosebased amphiphiles), 10Ac and 10 (lactose-based compound) and 12 (mélibiose) were not toxic at this concentration. Interestingly, the most active compound ohmline (10; 1-O-hexadecyl-2-O-methyl-racglycero-3-β-lactose) was not toxic at 10 μM. It was not possible to determine the toxic IC50 for ohmline, owing to its low water solubility that restricted the concentrations that could be tested. This limited water solubility imposed to dissolve the amphiphiles in a mixture of DMSO/ ethanol before a subsequent dilution in water or salt solutions. Importantly, this latter aqueous dilution reduced the concentration of DMSO and ethanol at a level which was much below the in vivo toxic cut-off of these solvents when administrated alone by intravenous route (Strickley, 2004). Indeed, for in vivo experiments ohmline was tested at 15 mg/kg that corresponds to 0.45 mg ohmline in 100 μL salt solution

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(6.87 mM in NaCl 0.9%) with 2% ethanol and 3% DMSO (Chantome et al., 2013). Additional in vitro studies revealed that the reduction of cell migration by ohmline was dependent on the SK3 channel for concentrations below 300 nM (Girault et al., 2011) to as low as 10 nM (lower concentrations were not tested) (Girault et al., 2011). However, at higher concentration (e.g. 1 μM), the reduction of cell migration was still observed, but at this concentration the reduction was not purely SK3-dependent since a reduction of cell migration was observed at this concentration in cells that do not express the SK3 channel (Girault et al., 2011). The question of the influence of the stereochemistry at the sn2 position of the glycerol moiety was also addressed. The R and S diastereoisomers of ohmline ((2R)-1-O-hexadecyl-2-O-methyl-sn-glycero-3-β-lactose and (2S)-1-O-hexadecyl-2-O-methyl-sn-glycero-3-β-lactose) were synthesized. Their evaluation (patch clamp measurements) revealed identical activities leading to the conclusion that the stereochemistry at the sn2 position of the glycerol moiety was not a determinant structural factor to modulate SK3 current (Girault et al., 2011). A second important aspect that was studied was the selectivity of action of ohmline with respect to other ion channels. This study was not undertaken with all the compounds but only with those exhibiting important action on SK3 ion channel, particularly ion channels belonging to the SKCa channel family. Ohmline was active on the SK3 channel (73% reduction of activity), weak on the SK1 channel (20% reduction of activity) and no effect was observed, neither on the SK2 channel nor on the IKCa/SK4 channel at 10 μM. The second set of variations of the structure of ohmline aimed at investigating the effect of the incorporation of one negative charge in the structure of the polar head. This structural modification aimed to slightly improve the water solubility of the new analogues. For this purpose, a phosphate moiety was included between the saccharide unit and the glycerol–ether lipid fragment as shown in Fig. 3 (Sevrain et al., 2012). It is important to note that in this series of molecules, the trimethylammonium fragment of the phosphocholine polar head of edelfosine was replaced with a neutral disaccharide moiety. For one of the compounds (14), the glycosylation was further optimized in order to produce two samples: one as a pure α anomer (α anomer refers herein to the chirality of acetal carbon of the saccharid unit bonded to glycerol moiety) form 14b (1-O-hexadecyl-2-O-methylrac-glycero-3-phospho-α-maltose) and a second with a mixture of α/β anomers 14a (1-O-hexadecyl-2-O-methyl-rac-glycero-3-phospho-α/βmaltose). The comparison of their effect on cancer cell migration and the measurement of the SK3 ion current modulation indicated that the α anomer (14b) was only slightly more efficient than the mixture of anomers 14a (Table 3). Interestingly, the incorporation of the negatively charged phosphate group between the saccharide unit and the glycerol moiety produced indeed more water soluble compounds. For example, compound 13, which is the phosphate analogue of ohmline, was more soluble in water than ohmline with upper limits of solubility in water of 13 mg/mL for compound 13 (1-O-hexadecyl-2-O-methyl-racglycero-3-phospho-α/β-maltose and 6 mg/mL for ohmline. However, the incorporation of a phosphate group in the structure of ohmline induced the reduction of its selectivity toward the SK3 channel (unpublished results). These results indicate that the structure of ohmline can be further modified to produce SK3 channel modulators. The incorporation of the phosphate group in this series of compounds (13–15) did not lead to an increase of in vitro toxicity (MTT tests revealed an absence of toxicity at 10 μM). This result contrasts with the toxicity of edelfosine (toxic at 10 μM) which featured a zwitterionic polar head instead of a phospho-saccharidic moiety. As shown in Table 2, ohmline but also the melibiose-based analogues presented interesting capacities to modulate SK3-dependent cell migration. The common point between ohmline (1-O-hexadecyl2-O-methyl-rac-glycero-3-β-lactose 10) and compound 12 (1-Ohexadecyl-2-O-methyl-rac-glycero-3-β-melibiose) is the presence of

Please cite this article as: Jaffrès, P.-A., et al., Alkyl ether lipids, ion channels and lipid raft reorganization in cancer therapy, Pharmacology & Therapeutics (2016), http://dx.doi.org/10.1016/j.pharmthera.2016.06.003

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Fig. 3. Chemical structure of GPGEL (glyco–phosphate–glycero–ether lipid). The diastereoisomeric purity of synthesized compounds is indicated by the ratio of α to β anomers.

one galactopyranosyl and one glucopyranosyl moieties but with a different connection (1 → 4 in the case of the lactose moiety present in ohmline and a 1 → 6 junction present in the melibiose group present in compound 12). To further explore new analogues of ohmline in a structure–activity relationship study, the effect of the structure of the disaccharide unit on the modulation of the SK3 channel was studied. As mentioned above, oat is able to replace plasma membrane phospho– glycero–lipids by glyco–glycero–lipids when it is grown under phosphorus deprivation (Andersson et al., 2005). More specifically, the glyco-lipid produced is mainly di-galactosyl-diacyl glycerol (DGDG; 1,2-di-O-acyl-sn-glycerol-3-O-β-(1 → 6)di-galactopyranose) in which the saccharide moiety features an α-galactopyranosyl-(1 → 6)-βgalactopyranosyl group. The replacement of the lactosyl moiety of ohmline by a (1 → 6) digalactosyl group required the construction of the disaccharide moiety which is not available as a pre-constructed building unit. A new methodology based on the use of trimethylsilyl as protecting group was adapted to produce compound 16 (1-Ohexadecyl-2-O-methyl-rac-glycero-3-O-(α-galactopyranosyl-(1 → 6)β-galactopyranose)) in 14 synthetic steps (Fig. 4) (Sevrain et al., 2013). The evaluation of this compound indicated that it could reduce SK3 current by 28.2% at 5 μM and SK3-dependent cell migration by 19.6% at 300 nM (Table 4). These inhibitory capacities, which are significantly below those of ohmline or melibiose-based compound 12, indicate that the activity is sensitive not only to the connection of the Table 3 Effect of GPGEL (glyco–phosphate–glycero–ether lipid) on SK3 current and SK3dependent cancer cell migration.

13 14a 14b 15

% of SK3 current modulation at 10 μMa

% of inhibition of cell migration at 100 nMb

−69.25 ± 4.32% (n = 4) −60.00 ± 11.00% (n = 5) −70.75 ± 8.52% (n = 4) −65.50 ± 6.90% (n = 4)

55.0 ± 0.8% (n = 3) 64.0 ± 2.3% (n = 3) 51.0 ± 0.8% (n = 3) 50.0 ± 1.4% (n = 3)

a Acute test: current recorded at 0 mV in HEK-293 cells expressing the recombinant SK3 channel (ratio of the current in the presence of tested compound to the current without the compound). b Chronic test: reduction of MDA-MB-435s cell migration at 100 nM for 24 h (% determined as the ratio of the number of cells that have migrated in the presence of tested compound to the number of cells that have migrated without the compound). The SK3dependent part of MDA-MB-435s cell migration is 60%.

disaccharide moieties (1 → 4 versus 1 → 6) but also to the nature of the saccharide units that constitute the disaccharide unit. Compound 16 was nontoxic (as determined by MTT assay) at 1 μM, but some toxicity appeared at 10 μM. More recently, we prepared an analogue of compound 16 that features a α-glucopyranosyl-(1 → 6)-β-galactopyranose dissacharide moiety. This compound 17 (1-O-hexadecyl-2-O-methylrac-glycero-3-O-(α-glucopyranosyl-(1→6)-β-galactopyranose; Fig. 4) is actually an epimer of compound 16. This compound 17, which was first tested in patch clamp measurement (acute test at 10 μM), exhibited similar modulation efficacy of SK3 to that of the benchmark compound ohmline (Berthe et al., 2016). The chronic test (evaluation of the modulation of cell migration at 300 nM for 24 h) confirmed these good results (50% of reduction of cell migration). Despite the lower efficiencies of ohmline analogues possessing a 1 → 6 disaccharide unit, further investigations must be carried out in order to better understand the relation between the structure of the amphiphiles and their capacity to modulate the SK3 channel. Such type of information also contributes to better understand the mechanism of action involved in the interaction of amphiphiles with bio-membrane lipids and bio-membrane embedded proteins. Further details in relation with the mechanism of action are presented below. 3. Mechanism of action of edelfosine and ohmline Unlike most of antitumor drugs, edelfosine and other AELs act through their interaction with cell membranes. Edelfosine was reported as the first anticancer drug acting through its interaction with lipid raft domains in cell membranes (Gajate & Mollinedo, 2001; Gajate et al., 2004; Gajate & Mollinedo, 2007), promoting their reorganization that eventually led to the triggering of apoptosis (see Section 4). In addition, the endoplasmic reticulum and mitochondria have also been reported to be involved in the mechanism of action of edelfosine (Gajate & Mollinedo, 2014). Edelfosine accumulates in the endoplasmic reticulum of a number of cancer cells, leading to apoptosis through an endoplasmic reticulum stress response (Nieto-Miguel et al., 2006, 2007; Gajate et al., 2012; Bonilla et al., 2015). In this regard, and because plasma membrane–endoplasmic reticulum contact sites regulate phosphatidylcholine synthesis (Tavassoli et al., 2013), it is worthwhile to note that edelfosine and other AEL related analogues, such as miltefosine, inhibit phosphatidylcholine synthesis in a number of tumor cells by inhibition

Please cite this article as: Jaffrès, P.-A., et al., Alkyl ether lipids, ion channels and lipid raft reorganization in cancer therapy, Pharmacology & Therapeutics (2016), http://dx.doi.org/10.1016/j.pharmthera.2016.06.003

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Fig. 4. Chemical structure of 1 → 6 digalactosyl (DG) analogue of ohmline.

of CTP:phosphocholine cytidylyiltransferase (CCT), the enzyme catalyzing the rate-limiting step in de novo phosphatidylcholine synthesis, by inhibiting its translocation from cytosol to membrane (Tronchere et al., 1991; Boggs et al., 1995; Cui et al., 1996; Baburina & Jackowski, 1998; Nieto-Miguel et al., 2006, 2007; Gajate et al., 2012; Bonilla et al., 2015). Defects and inhibition in phosphatidylcholine synthesis might trigger apoptosis and endoplasmic reticulum stress (Tronchere et al., 1991; Cui et al., 1996; Ramos et al., 2002; van der Sanden et al., 2003; Nieto-Miguel et al., 2006, 2007; Gajate et al., 2012; Bonilla et al., 2015). Edelfosine has also been found to accumulate in both lipid rafts and endoplasmic reticulum in Saccharomyces cerevisiae yeast (Zaremberg et al., 2005; Cuesta-Marban et al., 2013; Czyz et al., 2013). The evidence reported so far seems to suggest that edelfosine targets at least two major subcellular structures in a cell type-dependent manner, namely cell surface lipid rafts mainly in hematological cancer cells and endoplasmic reticulum mainly in solid tumor cells (Nieto-Miguel et al., 2006; Gajate & Mollinedo, 2014). Irrespective of the major role of membrane raft platforms and endoplasmic reticulum in edelfosine-mediated apoptosis, mitochondria and mitochondria-mediated signaling are critical in the eventual cell death outcome triggered by edelfosine (Gajate et al., 2000b; Gajate & Mollinedo, 2007; Nieto-Miguel et al., 2007; Gajate et al., 2012). Overexpression of Bcl-2 or Bcl-xL, which protects mitochondria, blocks edelfosine-induced apoptosis (Mollinedo et al., 1997; Gajate et al., 2000b; Gajate & Mollinedo, 2007; Gajate et al., 2012). Edelfosine has also been reported to co-localize with mitochondria, promoting an increase in mitochondrial membrane permeability (Mollinedo et al., 2011). This direct interaction of edelfosine with mitochondria could eventually lead to mitochondrial dysfunction and apoptosis (Mollinedo et al., 2011). Interestingly, edelfosine promoted a redistribution of lipid rafts from the plasma membrane to mitochondria, suggesting a

Table 4 Effect of DG analog of ohmline on SK3 current and SK3-dependent cancer cell migration.

16 a

% of SK3 current modulation at 5 μMa

% of inhibition of cell migration at 300 nMb

28.2 ± 2.0%

19.6 ± 2.7%

Acute test: current recorded at 0 mV in HEK-293 cells expressing the recombinant SK3 channel (ratio of the current in the presence of 5 μM compound 16 to the current without compound 16). b Chronic test: reduction of MDA-MB-435s cell migration at 300 nM for 24 h (% determined as the ratio of the number of cells that have migrated in the presence of tested compound to the number of cells that have migrated without the compound). The SK3dependent part of MDA-MB-435s cell migration is 60%.

raft-mediated link between plasma membrane and mitochondria (Mollinedo et al., 2011). Regarding signaling mechanisms, two major routes have been involved in edelfosine-induced apoptosis, namely: a) JNK activation and c-jun proto-oncogene expression (Mollinedo et al., 1994; Gajate et al., 1998; Ruiter et al., 1999; Gajate et al., 2012; Bonilla et al., 2015), and b) Akt signaling inhibition (Ruiter et al., 2003; Reis-Sobreiro et al., 2013). Edelfosine activates JNK through its recruitment into rafts (Gajate et al., 2004; Nieto-Miguel et al., 2008) and endoplasmic reticulum stress response (Nieto-Miguel et al., 2007; Gajate et al., 2012). On the other hand, it is worth to point out that inhibition of Akt, a serine/ threonine kinase acting downstream of the phosphatidylinositol 3′-kinase (PI3K)/PTEN signaling pathway, has been reported as one of the best characterized targets of AELs and related compounds, particularly the alkylphosphocholine perifosine (Kondapaka et al., 2003; Ruiter et al., 2003). In fact, Akt inhibition underlies the molecular basis for the use of perifosine in clinical trials (Richardson et al., 2012; Orlowski, 2013). Perifosine blocks Akt plasma membrane localization, thus preventing its activation (Kondapaka et al., 2003). In this regard, edelfosine and perifosine have been recently reported to displace Akt as well as regulatory proteins from lipid rafts (Reis-Sobreiro et al., 2013). Furthermore, perifosine, as well as edelfosine, have also been reported to act through recruitment of death receptors and downstream signaling molecules in lipid rafts (Gajate & Mollinedo, 2007). As mentioned above, because of their lipid nature, AELs can affect membrane proteins that are embedded into cell membranes such as ion channels. Edelfosine was found to act on membrane transporters and on ion channels that govern the influx and efflux of ions and nutrients. For example, edelfosine was found to inhibit Na+/H+ exchanger (Besson et al., 1996), Na+/K+ ATPase (Zheng et al., 1990) and to affect Ca2+-ATPase (Grosman, 2001). Additionally, edelfosine increased intracellular calcium concentration in normal and tumor cells by activating PAF receptors, leading to a store-operated calcium entry (Alonso et al., 1997; Jan et al., 1999). The direct effect of edelfosine on storeoperated channels (Orai/TRPC/STIM) that govern calcium entry following endoplasmic reticulum calcium release remains to be explored. Surprisingly, only a few experiments have tested the effect of edelfosine on ion channels despite their well-known regulation by phospholipids and lysophospholipids, including PAF and lysoPAF. Among them, the mechano-gated 2P domain potassium TREK-1 and TRAAK channels were found to be activated by lysoPAF. Edelfosine and ohmline were found to inhibit the SK3 channel leading to a reduction of cancer cell migration (Girault et al., 2011; Potier et al., 2011; Chantome et al., 2013). This effect is dose-dependent and with a selective inhibition of SK3-

Please cite this article as: Jaffrès, P.-A., et al., Alkyl ether lipids, ion channels and lipid raft reorganization in cancer therapy, Pharmacology & Therapeutics (2016), http://dx.doi.org/10.1016/j.pharmthera.2016.06.003

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dependent cell migration below 1 μM. The suppression of SK3 reduced cancer cell migration while the enforced expression of SK3 protein in cells that do not naturally express the channel (either cancer cells or non-cancer cells) increased their capacity to migrate. This channel is expressed in some cell types (e.g., brain, smooth muscle) and participates in cell excitability by controlling cellular secretion and muscle contraction. When SK3 protein is expressed in cancer cells, it increases two-fold the capacity of cells to migrate and invade a matrix similar to the physiological extracellular matrix. Thus, cancer cells seem to hijack the physiological function of the SK3 channel found in excitable cells to promote their ability to migrate. This channel is expressed as a complex with the Orai1 calcium channel in caveolae lipid rafts (Chantome et al., 2013). This complex was not observed in several non-tumor cells and seems specific to cancer cells (Gueguinou et al., 2014). Because of this novel and non-physiological function of SK3 channel, a role of the SK3 channel was hypothesized in the formation of metastases. Thus, using mouse models of metastatic breast cancers (orthotopic xenografts), the SK3 channel was found to promote the development of metastases, mainly located in bones — where there is a direct link between the activation of the SK3 channel by calcium and the high calcium concentrations found in the bone environment (Chantome et al., 2013). Edelfosine and ohmline reduced SK3 currents in cancer cells without affecting the expression of SK3 protein (Girault et al., 2011; Potier et al., 2011). In contrast to well established pore blockers, such as apamin, edelfosine and ohmline act as inhibitory lipids interacting at a site distinct from the apamin binding site in SK3 channel. Interestingly, edelfosine and ohmline were found to have no effect on the IKCa/SK4 channel, indicating a selectivity of these lipids toward the other members of the KCa channels. In addition, the potency of ohmline was greater for the SK3 channel than for the SK1 channel and it did not affect the SK2 channel (Girault et al., 2011). To investigate whether ohmline could interact with L-type voltage-gated calcium channel (at its dihydropyridine site), hERG channel or voltage-gated sodium channel, antagonist radioligand binding studies were performed. Ohmline did not inhibit 3H–nitrendipine binding, 3H–astemizole binding or 3H– batrachotoxin binding demonstrating that this lipid does not interact with these channel antagonist sites (Girault et al., 2011). Similar results were obtained with edelfosine (unpublished observation). These experiments did not rule out the possibility that edelfosine could act at a site that is distinct from the binding sites tested. Edelfosine reduced the intracellular calcium concentration of cancer cells, an effect that was not due to complexation of calcium by the phosphate group of edelfosine, but rather by the depolarization induced by its inhibitory effect on the SK3 channel and its probable effect on calcium sensitivity (Potier et al., 2011). In addition, edelfosine affected endoplasmic reticulumstored calcium level (Nieto-Miguel et al., 2007; Gajate et al., 2012). Ohmline and compound 17 (which does not have a phosphate group) also depolarized the plasma membrane but also delocalized the SK3 and Orai1 channels from caveolin fraction and reduced the constitutive calcium entry observed in cancer cells (Girault et al., 2011; Chantome et al., 2013; Berthe et al., 2016). The molecular structures of edelfosine and ohmline resemble that of PAF and it was shown that edelfosine was a ligand for PAF receptor (Alonso et al., 1997; Jan et al., 1999; Girault et al., 2011) even though the affinity of the PAF receptor for edelfosine was estimated to be about 5000 times smaller than for the natural lipid PAF (Alonso et al., 1997). In contrast to edelfosine, ohmline was unable to interact with PAF receptor or with LPA (lysophosphatidic acid) receptor (Girault et al., 2011). Moreover and in contrast to edelfosine, ohmline did not affect PKC (protein kinase C, various isoforms) activities and only interacted with PLC (phospholipase C) at concentrations higher than those found for edelfosine (Girault et al., 2011). If the SK3 channel was found to specifically participate in the development of bone metastases, its inhibitor ohmline was found to reduce not only bone metastasis development but also lung and lymph node metastases (Chantome et al., 2013). Note that in these experiments ohmline was injected at the same time as cancer cells were being

grafted, indicating that this compound prevents metastasis development. Ohmline had no effect on primary tumor development; this was not surprising since the SK3 channel did not regulate primary tumor development (Chantome et al., 2013). Very recently the action of ohmline on colon cancer cells (HCT-116) was assessed (Gueguinou et al., 2016). We found that the SK3 channel was co-localized with two other calcium channels (TRPC1/Orai1) in caveolae–lipid raft nano-domains. The association of these three ion channels, which was triggered by phosphorylated STIM1, induced calcium entries (SOCE) that led to the increase of cell migration. Moreover, the activation of the EGF receptor of HCT-116 cell line induced a phosphorylation of Akt and subsequently a phosphorylation of STIM1. These results indicate for the first time that EGF induced via the Aktdependent phosphorylation of STIM1 an activation of SOCE. Simultaneously, the increase of phosphorylated Akt activates the Rac1/calpain that also increases cell migration. After pointing out this mechanism that involved two loops of activation of SOCE-dependent cell migration (1-STIM1/Orai1/RTPC1/SK3 and 2-Rac1/calpain) ohmline induces a delocalisation of SK3 (it moves away from the lipid raft) leading to reduce SOCE and SOCE-dependent cell migration. Moreover, ohmline reduced the phosphorylation of Akt activated by EGF. Both effects contribute to reduce SOCE-dependent cell migration. More surprisingly, we found that two monoclonal antibodies used in clinical treatments (cetuximab and panitumumab) induce either an increase of SOCEdependent cell migration (cetixumab) or a decrease of cell migration (panitumumab). Interestingly, the association of ohmline with the above antibodies prevents the increase of cell migration (cetuximab) or increases the reduction of cell migration (panitumumab). These results show for the first time that ohmline is a modifier of therapeutic monoclonal antibody action (Gueguinou et al., 2016). 4. Importance of lipid rafts for edelfosine and ohmline's antitumor effect Membrane nanodomains named lipid rafts, enriched in sterols and sphingolipids have been found to play a major role for the correct functioning of several cell survival signaling pathways (Simons & Toomre, 2000; Mollinedo & Gajate, 2015) as well as of transporters and ion channels (Pani & Singh, 2009; Best & Kamp, 2012; Mollinedo, 2012; Rosenhouse-Dantsker et al., 2012; Gueguinou et al., 2014), including the SK3 channel (Chantome et al., 2013). In addition, lipid rafts have also been found to mediate death receptor signaling favoring the triggering of apoptosis (Gajate et al., 2004; Mollinedo & Gajate, 2006, 2010a; Gajate & Mollinedo, 2015b). Thus, lipid rafts are able to act as scaffolds for cell survival and cell death promoting machineries, highlighting their critical role in regulating cell fate. Edelfosine has been found to accumulate in lipid rafts in different cancer cell types (van der Luit et al., 2002; Gajate et al., 2004; Ausili et al., 2008; Gajate et al., 2009a; Mollinedo et al., 2010a; Cuesta-Marban et al., 2013) and modulate cell fate through either recruiting Fas/CD95 and additional death receptors into lipid rafts (Gajate & Mollinedo, 2001; Gajate et al., 2004; Gajate & Mollinedo, 2007), or by displacing Akt survival signaling in mammalian cells (Reis-Sobreiro et al., 2013) or the proton pumping ATPase Pma1p and additional raft-associated transporters in S. cerevisiae yeast (Zaremberg et al., 2005; Cuesta-Marban et al., 2013; Czyz et al., 2013) from rafts, which eventually lead to cell death. Edelfosine induced recruitment and clustering of Fas/CD95 death receptor as well as of FADD and procaspase-8/-10 in raft domains, thus facilitating the formation of the death-inducing signaling complex (DISC), required for triggering the early stages of death receptor-mediated apoptosis (Gajate & Mollinedo, 2007; Gajate et al., 2009a). In addition, edelfosine has been reported to induce recruitment into lipid rafts of downstream signaling molecules that eventually promote the onset of apoptosis, including c-Jun N-terminal kinase (JNK) and Bid (Gajate et al., 2004; Gajate & Mollinedo, 2007), this latter linking the death

Please cite this article as: Jaffrès, P.-A., et al., Alkyl ether lipids, ion channels and lipid raft reorganization in cancer therapy, Pharmacology & Therapeutics (2016), http://dx.doi.org/10.1016/j.pharmthera.2016.06.003

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receptor-mediated signaling pathway with the mitochondria-mediated signaling route (Luo et al., 1998; Gajate et al., 2009b; Mollinedo et al., 2011). Edelfosine treatment also recruited Hsp90, JNK and apoptotic molecules in lipid rafts, but not the survival signaling molecules extracellular signal-regulated kinase (ERK) and Akt (Nieto-Miguel et al., 2008). In this new lipid raft location, JNK is being associated with, either

9

directly or indirectly, and regulated by Hsp90. Because Hsp90 associates with JNK when both molecules are concentrated in lipid rafts, identifying JNK as a novel Hsp90 client protein, this suggests that Hsp90 is rather promiscuous in its interactions, depending on the scenario where it is located (Nieto-Miguel et al., 2008). Taken together, this recruitment and concentration of apoptotic molecules (Fig. 5), kept apart from survival

Fig. 5. Proposed mechanism of action of AELs in tumor cells. SK3 and Orai1 channels are embedded within lipid rafts and form a complex regulating cancer cell migration and metastasis development. The supramolecular entity CASMER is not formed in this condition and cells are resistant to apoptosis. The reorganization of lipid raft induced by edelfosine and ohmline allows Orai1–SK3 to move away from lipid rafts and abolishes SK3-dependent constitutive calcium entry. Thus, SK3-dependent cancer cell migration and bone metastases are counteracted. Edelfosine recruits a number of apoptotic signaling molecules in lipid rafts to generate the CASMER favoring the generation and amplification of apoptotic signals.

Please cite this article as: Jaffrès, P.-A., et al., Alkyl ether lipids, ion channels and lipid raft reorganization in cancer therapy, Pharmacology & Therapeutics (2016), http://dx.doi.org/10.1016/j.pharmthera.2016.06.003

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signaling molecules, in a rather small membrane area of lipid raft platforms, leads to a dramatic local change in the apoptosis/survival signals ratio in a specific subcellular structure that eventually triggers a cell death response (Nieto-Miguel et al., 2008; Mollinedo & Gajate, 2010a, 2010b, 2015; Gajate & Mollinedo, 2015b). Additional structurally related compounds, collectively known as alkylphospholipid analogues that keep structural relationship with the AEL edelfosine, such as perifosine, also promoted clustering of Fas/CD95 and downstream signaling molecules in lipid rafts, leading to apoptosis in cancer cells (Gajate & Mollinedo, 2007). This recruitment of Fas/CD95 in lipid rafts, initially reported following the treatment of cancer cells with edelfosine, has subsequently been observed to occur to a greater or lesser extent with several drugs showing anticancer activity (Table 5). This concentration of apoptotic signaling molecules in raft platforms led to the formation of a supramolecular structure coined CASMER (cluster of apoptotic signaling molecule-enriched rafts) as a raft membrane platform containing aggregated death receptors and apoptotic downstream signaling molecules, thus facilitating protein–protein interactions and the triggering of apoptosis (Fig. 5) (Mollinedo & Gajate, 2010a, 2010b; Gajate & Mollinedo, 2015b). In vitro and in vivo approaches indicate that lipid rafts could constitute a novel and promising target in cancer therapy (Gajate & Mollinedo, 2007; Mollinedo et al., 2010a, 2010b). The affinity of edelfosine for cholesterol and lipid rafts (Ausili et al., 2008; Busto et al., 2008), altering the biophysical features of lipid raft domains in model membranes, increasing their thickness and fluidity (Ausili et al., 2008; Castro et al., 2013), and leading to the reorganization of these raft domains following edelfosine treatment, suggests that this AEL could constitute a paradigmatic and lead compound in this novel raftmediated therapy (Mollinedo et al., 2010a; Gajate & Mollinedo, 2011, 2014, 2015b). The above data indicate that AELs promote translocation of death receptors and apoptotic signaling molecules that eventually turn on a cell demise program. However, edelfosine has also been shown to inhibit Akt by displacing survival Akt signaling pathway from lipid rafts in mantle cell lymphoma cells (Reis-Sobreiro et al., 2013). Taken together,

the current evidence suggests that edelfosine can induce cell death in cancer cells by recruiting apoptotic molecules in lipid rafts and displacing survival molecules from these membrane domains (Gajate & Mollinedo, 2015a). In this regard, recent data have shown that lipid rafts are also involved in the mechanism of action of the AEL ohmline (unpublished observations). Ohmline acted as a disrupting agent for SK3–Orai1 lipid raft localization and upon ohmline treatment, the SK3–Orai1 complex moved away from lipid rafts (Fig. 5), and the SK3-dependent calcium entry was abolished (Chantome et al., 2013). Interestingly, ohmline was found in caveolae-enriched membrane fractions, where the SK3 channel was located following ultracentrifugation membrane fractionation, and when the SK3 channel was not localized in caveolae, ohmline had no effect on the SK3 channel (unpublished observations). The mechanism of action of ohmline on the SK3 channel is currently not known but a few hypotheses seem promising. A main hypothesis leading to a global scenario is that ohmline efficiency is associated to a modification of the biophysical properties of the plasma membrane related to its amphiphilic nature and inverted-conical shape. Because of its amphiphilic nature, insertion of ohmline into the lipid membrane would affect the activity of lipid-sensitive channels, possibly by changing membrane fluidity. It is interesting to mention that plasma membranes of cancer cells are more fluid compared to non-tumor cells (Plodinec et al., 2012). Higher membrane fluidity on cancer cells closely relates to their invasive potential, proliferation, and metastatic ability. Targeting of membrane fluidity, a biophysical characteristic of cell and normalization of membrane fluidity in cancer cells may represent a novel therapeutic modality (Swaminathan et al., 2011; Garg et al., 2015). Molecular dynamic simulation suggests that the lack of carbonyl group in AELs favors the cholesterol OH–phosphate interaction (Pan et al., 2012). This rearrangement would lead to a less hydrated final state interface, modifying the fluidity and probably the availability of cholesterol for specific domains in plasma membranes. It was already observed that lipids can modulate ion channels because of their geometric shape. Indeed, the inverted-conical shape of lysophospholipids such as lysophosphatidylcholine (LPC) as well as PAF (independently of its receptor) tends to favor a convex deformation

Table 5 Recruitment of death receptors and downstream signaling molecules into lipid rafts by anticancer drugs and signaling-modulating agents in cancer cells. CEM, human acute T-cell leukemia cell line; HT29, human colon carcinoma cell line; Jurkat, human acute T-cell leukemia cell line; M624, human melanoma cell line; MM144, human multiple myeloma cell line; Mz-ChA-1 cells, human cholangiocarcinoma cell line; PC-3, human prostate cancer cell line; SW480, human colon carcinoma cell line; Ramos, human Burkitt's lymphoma cell line; SNU601, human gastric cancer cell line; SNU601/R, cisplatin-resistant SNU601 gastric cancer subline. DR4 (death receptor 4), also known as TRAIL receptor 1. DR5 (death receptor 5), also known as TRAIL receptor 2. Anticancer drug/ signaling modulation

Cancer cells

Death receptors (and downstream signaling molecules) recruited in rafts

References

Akt signaling inhibition (Akt inh-VIII) Anandamide Aplidin Avicin D Ceramidea Cisplatin Cryptocaryone Edelfosine

Jurkat

Fas/CD95

Pizon et al., 2011

Mz-ChA-1 Jurkat Jurkat Jurkat HT29 PC-3 Jurkat MM144

Perifosine PI3K signaling inhibition (LY294002, wortmannin) Resveratrol

MM144 Jurkat, CEM

Fas/CD95 Fas/CD95, DR5, TNF-R1 (FADD, procaspase-8, procaspase-10, JNK, Bid) Fas/CD95 (FADD, procaspase-8, procaspase-7, Bid) Fas/CD95 Fas/CD95 (FADD, procaspase-8) Fas/CD95, DR4, DR5 (FADD, procaspase-8) Fas/CD95 (FADD, procaspase-8, procaspase-10, JNK, Bid) Fas/CD95, DR4, DR5, TNF-R1 (FADD, procaspase-8, procaspase-9, procaspase-10, JNK, Bid, cytochrome c, APAF-1) Fas/CD95, DR4, DR5 (FADD, procaspase-8, Bid) Fas/CD95

DeMorrow et al., 2007 Gajate & Mollinedo, 2005 Xu et al., 2009 Cremesti et al., 2001; Grassme et al., 2001, 2003 Lacour et al., 2004 Chen et al., 2010 Gajate & Mollinedo, 2001; Gajate et al., 2004, 2009a Gajate & Mollinedo, 2007; Gajate et al., 2009b; Mollinedo et al., 2010a Gajate & Mollinedo, 2007 Beneteau et al., 2008; Pizon et al., 2011

Fas/CD95, DR4, DR5 (FADD, procaspase-8) Fas/CD95, DR5 (FADD, procaspase-8, procaspase-10, JNK, Bid) Fas/CD95, DR4, DR5 (FADD, procaspase-8, procaspase-10, JNK, Bid) CD95 (FADD, procaspase-8) Fas/CD95 (FADD, procaspase-8) Fas/CD95 (FADD, procaspase-8) Fas/CD95, DR5

Delmas et al., 2004 Reis-Sobreiro et al., 2009 Reis-Sobreiro et al., 2009 Delmas et al., 2003 Stel et al., 2007 Elyassaki & Wu, 2006 Lim & Han, 2015

Rituximab Ultraviolet light Ursodeoxycholic acid a

HT29 Jurkat MM144 SW480 Ramos M624 SNU601, SNU601/R

Acting not as an inducer, but as an amplifier of a previous triggering of Fas/CD95 response by its cognate ligand or agonistic antibodies.

Please cite this article as: Jaffrès, P.-A., et al., Alkyl ether lipids, ion channels and lipid raft reorganization in cancer therapy, Pharmacology & Therapeutics (2016), http://dx.doi.org/10.1016/j.pharmthera.2016.06.003

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of the plasma membrane, which led to mechano-gated 2P domain potassium channels TREK-1/TRAAK channels opening (Maingret et al., 2000; Patel et al., 2001). The cone shape hypothesis assumes that amphipathic compounds integrate mainly in one or the other leaflet of the phospholipid bilayer that is deformed in opposite directions by inverted-cone-shaped (edelfosine/ohmline) and cone-shaped lipids (e.g. phosphatidylethanolamine, PE). The effect of cone or inverted cone-shaped lipids as inhibitors of the SK3 channel needs to be tested. The conical shape hypothesis on TREK-1/TRAAK channels also implies that the potency of cone or inverted-cone structures is directly related to the size of the polar head (Maingret et al., 2000; Patel et al., 2001). It was observed that the suppression of the phosphocholine group suppressed the inhibitory effect of edelfosine on the SK3 channel and that the replacement of the lactose unit (disaccharide), present in ohmline, by a mono-saccharide moiety produced a less effective inhibitor of the SK3 channel (Girault et al., 2011). Lysophospholipids and PE have opposite conical shapes that lead, when they are present in the same bilayer, to a lipid global cylindrical organization that stabilizes the bilayer organization (Madden & Cullis, 1982). It is worth mentioning that recent data suggest that ohmline, unlike edelfosine (Ausili et al., 2008; Castro et al., 2013), rigidifies liposome and cell membrane (unpublished observations obtained using the fluorescent probe Laurdan that senses the fluidity of the lipid bilayer). Maybe differences on the polar group between edelfosine and ohmline might explain this apparent difference. It was already mentioned above that the higher membrane fluidity in cancer cells is related to their invasive potential. For instance, genistein exerts its antimetastatic effects by changing the mechanical (fluidity) properties of prostate cancer cells (Ajdzanovic et al., 2013). Membrane fluidity, determined by steadystate fluorescence polarization measurements, was correlated with the metastatic capacity of murine tumor-cell lines, which is regulated by membrane lipid composition or culture conditions. These results might contribute to explain the role of membrane fluidity observed in cancer metastasis (Taraboletti et al., 1989). Another hypothesis is that ohmline would directly interact with the SK3 channel. This hypothesis would partly or fully explain the selectivity of action of ohmline for the SK3 channel and also its dose–effect relationship that has been reported for both acute tests (patch clamp experiments) and chronic tests (SK3-dependent cell migration). Of note, interaction between amphiphilic compounds and potassium channels including the SK2 channel (a member of the SKCa family as SK3) was previously reported (direct interaction between the amphiphile lipid phosphatidylinositol 4,5-diphosphate PIP2 and the Kir2.2 and SK2 potassium channel) (Huang et al., 1998; Zhang et al., 2015), suggesting that such a specific interaction could also occur between the ohmline and the SK3 channel. 5. Clinical therapeutic use of edelfosine, alkylphosphocholines, and ohmline in cancer 5.1. Edelfosine Preclinical studies have shown that edelfosine is an efficient antitumor drug in several mouse xenograft models (Mollinedo et al., 2010a, 2010b; Gajate et al., 2012; Bonilla et al., 2015). In addition, edelfosine accumulates preferentially in tumor cells in both in vitro (Mollinedo et al., 1997; Gajate et al., 2000a, 2004; Gajate & Mollinedo, 2007) and in vivo studies (Estella-Hermoso de Mendoza et al., 2009; Mollinedo et al., 2010a, 2010b). Edelfosine has been shown to inhibit metastasis (Estella-Hermoso de Mendoza et al., 2012) and angiogenesis (Candal et al., 1994; Vogler et al., 1998). The antimetastatic activity of edelfosine is increased by the use of edelfosine-containing lipid nanoparticles (Estella-Hermoso de Mendoza et al., 2012). Edelfosine shows a rather selective action against tumor cells (Mollinedo et al., 1997; Gajate et al., 2000a, 2004; Gajate & Mollinedo, 2007; Mollinedo et al., 2010a, 2010b), has no apparent toxicity to vital

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organs, without any significant cardiotoxicity, hepatotoxicity or renal toxicity, and is a non-myelosuppressive agent (Gajate & Mollinedo, 2002; Mollinedo et al., 2009). These features underlie the fact that purging in autologous bone marrow transplantation, in leukemia and other cancers, is so far the major indication for a clinical use of edelfosine as exhaustively detailed by Gajate and Mollinedo in a previous review (Gajate & Mollinedo, 2002). However, a phase II clinical trial of edelfosine alone with non-small cell lung carcinoma (NSCL) was rather disappointing (Gajate & Mollinedo, 2002). Now, our better understanding of Erufosine, ErPC3 the mechanisms of action of edelfosine should enable clinicians to better position this molecule in the clinic and to discern which tumors could be more suitable for AEL therapy. During the last years a number of studies have shown that edelfosine is active against a wide array of different tumor types, including multiple myeloma (Gajate & Mollinedo, 2007; Mollinedo et al., 2010a), mantle cell lymphoma, chronic lymphocytic leukemia (Mollinedo et al., 2010b), Ewing sarcoma (Bonilla et al., 2015), pancreatic cancer (Gajate et al., 2012), and glioma (Melo-Lima et al., 2015b). Because of the ability of edelfosine to recruit death receptors in raft platforms, this AEL potentiates the action of death ligands, including FasL/CD95L and TRAIL or agonistic anti-death receptor antibodies in human myeloma cells (Gajate & Mollinedo, 2007). This could be of particular interest, as TRAIL shows a selective and promising antitumor action in different tumor cells (Mitsiades et al., 2001; Yagita et al., 2004; Lim et al., 2015). Several reports have shown an increase in the cytotoxic activity of edelfosine when combined with additional antitumor drugs, such as: a) vincristine in human glioma cells (Haugland et al., 1999); b) merocyanine 540 in human neuroblastoma, osteosarcoma, Wilms' tumor, leukemia, breast cancer cells, as well as murine neuroblastoma and breast cancer cells (Yamazaki & Sieber, 1997; Anderson et al., 2002, 2003); and c) gemcitabine with clofarabine in human T cell-lymphoma cells (Valdez et al., 2014). 5.2. Alkylphosphocholines The synthesis in the late 80s of miltefosine (hexadecylphosphocholine) (Fig. 6), lacking the glycerol motif, led to the generation of a new subfamily of antitumor lipids, known as alkylphosphocholines, originally derived from lysophosphatidylcholine with a simplified structure (Gajate & Mollinedo, 2002; Mollinedo, 2007, 2014). Miltefosine is already used in the clinic (6% miltefosine solution under the trademark of Miltex®, ASTA Medica, later Æterna Zentaris GmbH, Frankfurt, Germany) as a palliative and topical treatment option for cutaneous metastases from breast cancer (Clive et al., 1999; Leonard et al., 2001). In addition, miltefosine has become the first oral drug, registered as Impavido® (Æterna Zentaris GmbH, Frankfurt, Germany; Paladin Therapeutics, Montreal, Canada), for the treatment of visceral leishmaniasis in several countries (Sundar et al., 2006; Berman, 2008; Dorlo et al., 2012). Later on in the late 90s perifosine was synthesized in an attempt to improve the antitumor therapeutic potency by replacing the choline moiety of miltefosine by a heterocyclic piperidine group (Hilgard et al., 1997) (Fig. 6). Targeting the pleckstrin homology domain of Akt is suggested to be the main mode of action of perifosine, thereby preventing its translocation to the plasma membrane and inhibiting the activation of the PI3K/Akt pathway (Kondapaka et al., 2003; Ruiter et al., 2003; Krawczyk et al., 2013). A high number of clinical trials have been conducted and oral administration of perifosine was generally well tolerated, with gastrointestinal and fatigue being the most common toxicities observed. However, no significant clinical activity against a number of cancers was reported in phase II clinical trials for different tumors, including: malignant melanoma (Ernst et al., 2005); prostate cancer (Posadas et al., 2005; Chee et al., 2007); metastatic or locally advanced soft tissue sarcoma (Knowling et al., 2006); recurrent or metastatic head and neck cancer (Argiris et al., 2006); advanced, unresectable, or metastatic pancreatic adenocarcinoma (Marsh Rde et al., 2007); and metastatic breast cancer (Leighl et al., 2008). Perifosine showed activity

Please cite this article as: Jaffrès, P.-A., et al., Alkyl ether lipids, ion channels and lipid raft reorganization in cancer therapy, Pharmacology & Therapeutics (2016), http://dx.doi.org/10.1016/j.pharmthera.2016.06.003

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Fig. 6. Chemical structures of some clinically relevant alkylphosphocholine analogues.

in patients with advanced renal cell carcinoma after failure on VEGFtargeted therapy, but its activity was not superior to currently available second-line agents (Cho et al., 2012). In general, the above studies suggested that perifosine did not show sufficient activity as a single agent to warrant further development. Nevertheless, despite not achieving the primary objectives related to significant increases in progression-free survival rates, some optimism still remained for this agent in advanced soft tissue sarcoma patients (Bailey et al., 2006), as well as to warrant further evaluation of perifosine in combination with rituximab or other active agents in patients with relapsed/refractory Waldenstrom's macroglobulinemia (Ghobrial et al., 2010), and with currently available therapies in renal cancer (Cho et al., 2012). Furthermore, a number of studies have reported the potentiation of antitumor activity following combination of perifosine with distinct anticancer drugs in cancer cells derived from several types of leukemia (Nyakern et al., 2006; Papa et al., 2008; Tazzari et al., 2008), multiple myeloma (Hideshima et al., 2006; Gajate & Mollinedo, 2007), osteosarcoma (Yao et al., 2013), medulloblastoma (Kumar et al., 2009), lung cancer (Elrod et al., 2007), colon cancer (Chen et al., 2012), and glioma (Momota et al., 2005), as well as following combination of miltefosine with different anticancer compounds or treatments in distinct cancer cell types (Haberkorn et al., 1992; Spruss et al., 1993; Papagiannaros et al., 2006; Thakur et al., 2013). Thus, phase II clinical trials were conducted with promising results using perifosine (KRX-0401) in combination with bortezomib (Velcade) and dexamethasone (Decadron) in multiple myeloma (Richardson et al., 2011) and in combination with capecitabine in metastatic colorectal cancer (Bendell et al., 2011). This led to recent phase III clinical studies, but unfortunately these latter failed to demonstrate

significant extension of progression-free survival in patients with relapsed and refractory multiple myeloma and metastatic colorectal cancer (Fensterle et al., 2014), compared to Velcade and dexamethasone alone (multiple myeloma), or to capecitabine (colorectal cancer) (Table 6). Nevertheless, additional combinations of perifosine with current therapeutic drugs are still in progress. In this regard, a phase II clinical trial using perifosine and sorafenib combination therapy showed some promising results with manageable toxicity in patients with Hodgkin lymphoma (Guidetti et al., 2014). In addition, a phase II clinical trial of perifosine and Torisel (temsirolimus) in patients with recurrent/ progressive malignant glioma to determine the induction of tumor shrinkage or stabilization and/or a delay in disease progression (NCT02238496) (Andrew Lassman, Columbia University; Pfizer; Æterna Zentaris) is underway. Thus, despite the initial failures in the above phase III trials (Table 6), the favorable safety and tolerability profiles of perifosine, together with the widely reported potentiation of the antitumor activity following combination of perifosine with other antitumor drugs and radiotherapy in in vitro and preclinical assays (reviewed in Krawczyk et al., 2013; Fensterle et al., 2014; Verheij et al., 2014), warrant additional well-designed clinical trials using combination therapy in the future. A description of the clinical trials of perifosine, either as a single agent or in combination therapy, has been previously summarized in several reviews (Gills & Dennis, 2009; Richardson et al., 2012; Krawczyk et al., 2013; Pachioni Jde et al., 2013; Verheij et al., 2014). Perifosine and other structurally related compounds have also been shown as potent radiosensitizers (Vink et al., 2006a, 2006b, 2007; Gao et al., 2011; Verheij et al., 2014), thus providing support for further

Table 6 Phase III clinical trials of perifosine in human cancers. Drugs

Type of cancer

Status

Perifosine + capecitabine

Metastatic and refractory Completed (April 2012). advanced colorectal cancer (X-PECT study)

Perifosine + Velcade (bortezomib) + dexamethasone (Decadron)

Relapsed and refractory multiple myeloma

National Clinical Observations Trial (NCT) identifier number NCT01097018

NCT01002248 Halted in March 2013. On March 11, 2013, an independent Data Safety Monitoring Board (“DSMB”) recommended that patient enrollment be stopped and the study discontinued.

Sponsor References

No benefit in overall survival Æterna Bendell et al., 2012 Zentaris Additional related references: Bendell et al., 2011; Richardson et al., 2012 Æterna Richardson et al., 2013 No significant extension of Zentaris Additional related progression-free survival references: Richardson compared to Velcade + et al., 2011, 2012; dexamethasone. No safety Orlowski, 2013 concerns were raised.

Please cite this article as: Jaffrès, P.-A., et al., Alkyl ether lipids, ion channels and lipid raft reorganization in cancer therapy, Pharmacology & Therapeutics (2016), http://dx.doi.org/10.1016/j.pharmthera.2016.06.003

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development of this combination therapy (radioptherapy with perifosine or additional alkylphosphocholine analogues) in both preclinical and clinical studies. Two of the latest derivatives of the synthetic alkylphosphocholine subfamily are erucylphosphocholine (ErPC) and erufosine (erucylphosphoN,N,N,-trimethylpropylammonium, ErPC3, erucylphosphohomocholine) with a 22-carbon chain (instead of a 16-carbon chain in miltefosine) and the presence of a cis-13,14 double bond (Fig. 6). Erufosine differs from erucylphosphocholine by the insertion of an additional methylene group into the polar phosphocholine choline head (Fig. 6). These rather small structural modifications increase hydrophobicity that contributes to the formation of lamellar structures, thus preventing hemolytic activity (Kaufmann-Kolle et al., 1996). This allows these new alkylphosphocholine analogues to be applied intravenously, leading to tumor responses comparable to oral administration, but at 5 times lower doses and with reduced gastrointestinal side effects. Erucylphosphocholine and erufosine have been shown to cross the blood–brain barrier and to accumulate at certain degree in the brain tissue (Erdlenbruch et al., 1999; Henke et al., 2009). These properties could support these alkylphosphocholine analogues as promising drugs for the treatment of brain tumors. Erufosine, similar to erucylphosphocholine, shows antitumor activity at the micromolar range and induces apoptosis in distinct types of tumor cells, including otherwise apoptosis-resistant glioblastoma cell lines (Fiegl et al., 2008; Yosifov et al., 2009; Konigs et al., 2010; Rudner et al., 2010; Bagley et al., 2011; Yosifov et al., 2011; Dineva et al., 2012; Kapoor et al., 2012; Kaleagasioglu & Berger, 2014). Erufosine interferes with survival signals (Chometon et al., 2014), and interacts with the 18 kDa mitochondrial translocator protein leading to activation of the mitochondrial apoptosis cascade (Veenman et al., 2014). The retinoblastoma signaling pathway has been shown to be essential for mediating the antitumor activity of erufosine, and hence its efficacy might be predicted by determining the retinoblastoma protein status (Zaharieva et al., 2007, 2014). Both in vitro and in vivo assays have shown promising antitumor activity of erufosine alone and in combination with radiation on astrocytoma and glioblastoma cell lines (Rubel et al., 2006; Awde et al., 2013). However, a preclinical in vivo study analyzing the effect of ionizing radiation in combination with erufosine on T98G glioblastoma xenograft tumors showed a transient decrease in the growth of the T98G tumors, but failed to improve efficacy of fractionated irradiation in terms of local tumor control (Henke et al., 2012). Some of the disappointing clinical outcomes described above do not exclude that these drugs could be effective in specific patient subgroups (Posadas et al., 2005). On the other hand, because low doses of the above drugs are well tolerated in patients, prolonged treatment schedules may be considered to achive a positive response for future experiments in in vivo studies (Henke et al., 2009). Interestingly, a number of studies have shown that edelfosine is the most active and potent inducer of apoptosis when compared to different alkylphosphocholine analogues in different types of cancer cells (Mollinedo et al., 1997, 2010b; Gajate et al., 2012; Bonilla et al., 2015). Thus, these data might suggest that the basic skeleton of the chemical structure of edelfosine remains as the most efficient one regarding apoptosis triggering. Because alkylphosphocholines include a drastic chemical change in the glycerol moiety of edelfosine, which is replaced by a long-chain alcohol conjugated to the phosphocholine head group or related polar group, additional analogues that keep the ether bonds in the glycerol backbone of the phospholipid but modify the head group could be worth to study and might lead to novel promising antitumor drugs, such as ohmline. 5.3. Ohmline Several non-phosphorus glycosidated ether lipids have been found to have an anti-tumoral activity (reviewed by (Arthur & Bittman, 2014)). The ability of these ether lipids to kill tumor cells independently of their action on mitochondria and via an apoptosis-independent

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mechanism makes the use of these compounds relevant against chemo-resistant tumors (Arthur & Bittman, 2014; Melo-Lima et al., 2015a). Ohmline does not show this cytotoxic effect and, because of its specific effect on the SK3 channel, it reduces cancer cell migration and metastasis development (see Section 3). Although ohmline was found to accumulate in all tumor tissues tested including in primary tumors, it preferentially reduced bone metastasis development and not the growth of the primary tumor (Chantome et al., 2013). Ohmline could prevent cancer cell migration to or in bones and thus bone colonization. Such a therapeutic approach might be of benefit to patients that are predisposed to develop bone metastases, mainly, according to incidence of bone metastasis in prostate, breast, lung and kidney cancers. The list of patents relating to the use of potassium channel modulators in cancer treatment is increasing (Villalonga et al., 2007). We have filed the first patent relating to the possible use of SK3 blockers in anticancer treatments (Potier et al., 2008), including alkyl-lipids for the prevention of metastasis (Bougnoux et al., 2011). Currently, to our knowledge, no treatment exists that can prevent or significantly reduce bone metastases, with the exception of Denosumab for which the results of a phase III clinical trial show a delayed onset of bone metastases of a few months (Smith et al., 2012). If the objective is the same, i.e. to reduce the development of cancer cells in the bone environment, the Denosumab monoclonal antibody against RANKL therapeutic approach differs radically from the anti-SK3 channel approach because Denosumab does not act on cancer cells but on the host tissue, the bone, where it prevents the lysis induced by the development of bone metastasis. Denosumab and Zoledronic acid, a bisphosphonate, are on the market for the prevention of skeletal-related-events in patients with solid tumors and bone metastases. Zoledronic acid suppresses bone breakdown by osteoclasts and similar to Denosumab, does not target cancer cells, thus differing from the SK3 channel approach. Recently (May 2013), a radiopharmaceutical, Radium 223 (Alpharadin) received marketing approval by the FDA as a treatment for castration-resistant prostate cancer in patients with bone metastases. It should be noted that in contrast to Denosumab and Zoledronic acid, Alpharadin demonstrated its ability to extend overall survival. Thus, Denosumab/Zoledronic acid/Alpharadin treatment approaches are complementary to anti-SK3 channel treatment and unlike Denosumab, Zoledronic acid and Alpharadin, the anti-SK3 channel therapeutic approach is targeted and specific, thus theoretically reducing adverse effects. 6. Administration route and formulations for the putative clinical use of edelfosine and ohmline A great advantage of edelfosine over other antitumor drugs lies in its solubility in water and aqueous solutions, and in the fact that it can be administered orally (Gajate & Mollinedo, 2002). This oral administration makes treatment with this drug safe, efficient and easily accessible, thus eliminating the need to visit hospital for intravenous therapy, like in many other antitumor drugs, or other medical procedures. The convenience and compliance benefits of oral treatment might encourage patients to remain on this therapy for longer periods. Furthermore, previous in vivo studies in rats have shown that edelfosine oral treatment showed no significant cardiotoxicity, hepatotoxicity or renal toxicity (Mollinedo et al., 2009). Phase I and II clinical trials with the so-called alkylphospholipid analogues that are structurally related to edelfosine, particularly perifosine, developed by Æterna Zentaris, Frankfurt, Germany, have shown a satisfactory safety and tolerability profile (Krawczyk et al., 2013; Fensterle et al., 2014). As mentioned above, miltefosine, the only alkylphospholipid analogue that is currently in the clinic, is used under the trademark of Miltex® for topical treatment of metastatic skin lesions in breast cancer (Leonard et al., 2001), and under the trademark of Impavido® as the first and still the only oral drug available for the treatment of leishmaniasis. Both topical and oral administration of miltefosine are well tolerated and only occasionally

Please cite this article as: Jaffrès, P.-A., et al., Alkyl ether lipids, ion channels and lipid raft reorganization in cancer therapy, Pharmacology & Therapeutics (2016), http://dx.doi.org/10.1016/j.pharmthera.2016.06.003

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require cessation of treatment (Dorlo et al., 2012). Miltefosine is also used under the trademark name of Milteforan® (Virbac, Carros, France) for the treatment of canine leishmaniasis with a low toxicity profile (Woerly et al., 2009). Previous clinical data gathered in preceding reviews have highlighted the particular low level of toxicity exhibited by edelfosine and its good tolerability profile (Munder & Westphal, 1990; Drings et al., 1992; Gajate & Mollinedo, 2002). Taken together, these preclinical and clinical data indicate that most frequent side effects are related to manageable gastrointestinal problems, and thereby toxicity is not a major issue of concern. The incorporation of edelfosine in either cholesterol-rich liposomal formulations (Busto et al., 2008) or lipid-based nanoparticles (Lasa-Saracibar et al., 2014), is a strategy to minimize its already low toxicity and to improve in vivo antitumor efficacy as well the pharmacodynamics and pharmacokinetics parameters (Estella-Hermoso de Mendoza et al., 2011, 2012). For ohmline, its low toxicity doesn't require to incorporate it in liposomal solution to avoid side effects. However, its limited water solubility renders its incorporation in either liposomal solutions or lipid nanoparticles attractive for the achievement of some in vivo studies. This work still remains to be done. 7. Clinical perspectives A series of clinical studies with edelfosine, including phase I and II clinical trials have been reported and discussed in detail in a previous review (Gajate & Mollinedo, 2002), indicating very low levels of toxicity and a favorable side effect profile. Likewise, perifosine has shown a favorable tolerability and side effect profile during phase I and II clinical trials (Krawczyk et al., 2013; Fensterle et al., 2014). However, as mentioned above, despite early encouraging results in phase II trials by using the combination of perifosine plus bortezomib and dexamethasone in patients with relapsed/refractory multiple myeloma (Richardson et al., 2011), and perifosine plus capecitabine in patients with metastasic colorectal cancer (Bendell et al., 2011), unfortunately these promising data were not borne out in further studies, thus failing to demonstrate sufficient clinical activity in recent phase III clinical trials (Fensterle et al., 2014). A detailed overview of clinical trials conducted with perifosine, used as a single agent or in combination with other anticancer treatments, has been recently reported (Verheij et al., 2014). A number of studies also show promise of perifosine as an attractive candidate as clinical radiosensitizer (Gao et al., 2011; Verheij et al., 2014). Because AELs and related compounds affect signaling routes that influence tumor cell radiosensitivity, they can be considered as promising agents to combine with radiotherapy (Vink et al., 2007; van Blitterswijk & Verheij, 2008; Verheij et al., 2014). Regarding edelfosine, its peculiar features suggest that this AEL could be an effective and well-tolerated drug for cancer treatment, causing minimal discomfort. However, it would be advisable to perform a number of additional preclinical and pharmacological studies in order to single out one or a few tumors for which the use of edelfosine, either alone or in combination with additional drugs, could lead to improved outcomes, before mounting a clinical trial with a high probability of success. In this regard, we are preparing novel formulations using new kinds of nanoparticles that, in preliminary studies, enhance the antitumor activity of the drug, as well as we are testing new edelfosinebased combination therapy regimens to avoid drug resistance and increase efficiency (unpublished observations). Unveiling the molecular mechanism of action of this drug is helping us to design better strategies to attack the tumor cell in an efficient and selective way. Interestingly enough, edelfosine shows a number of biomedical activities that make this drug rather unique in its versatility. Thus, edelfosine has been found to promote apoptosis in activated lymphocytes (Cabaner et al., 1999), hence suggesting a role for this drug in the treatment of autoimmune diseases (Mollinedo, 2007; Abramowski et al., 2014; Gajate & Mollinedo, 2015a). In addition, in vitro and in vivo assays in animal models have shown that edelfosine is a potent anti-inflammatory agent (Mollinedo et al., 2009), as well as an effective

drug against leishmaniasis (Varela et al., 2012, 2014) and schistosomiasis (Yepes et al., 2014, 2015). Additional biomedical applications and patents for AELs and edelfosine-related compounds have been previously reviewed (Mollinedo, 2007). On the other hand, we consider using the SK3 channel as a therapeutic target and ohmline for targeted therapy against bone metastasis development in patients with SK3-expressing tumor cells. The clinical relevance of the SK3 channel in cancer is ongoing. In patients, the analysis of a first series of samples of cancer with marked bone tropism (prostate, breast, kidney) showed that the SK3 channel was expressed in more than 60% of bone metastases. Ohmline has already passed the chemical optimization phase (Girault et al., 2011). Ohmline is well tolerated and is apparently safe in rodents with no observable tissue damage in rodents (Girault et al., 2011; Chantome et al., 2013). The preclinical studies performed in vitro have not shown any adverse biological activity and no toxicity or genotoxicity of ohmline at concentrations lower than 10 μM (Girault et al., 2011; Chantome et al., 2013). Finally, preliminary studies have been conducted and have evaluated the incorporation of ohmline in tissues or organs in the small rodent. These studies indicated that ohmline was incorporated in primary tumors and in bone metastasis tumors (Chantome et al., 2013). Non-metastatic prostate cancers with acquired characteristics of hormone-resistance represent a clinical situation adapted to the early investigation of ohmline. In these clinical settings, the probability of bone metastasis is elevated, and there is no alternative approach aimed at preventing the occurrence of metastases. Therefore, as soon as compelling preclinical data are available, ohmline could ethically be considered as an adjuvant treatment for the prevention of bone metastases. Using bone metastasis appearance as the endpoint, and assuming a lack of toxicity, the conditions would be brought together for proposing a randomized setting. This represents a rapid and efficient approach to investigate the antimetastatic potential of ohmline in prostate cancer. In case of success in prostate cancer, ohmline could be proposed for the prevention of bone metastases in cancers with marked bone tropisms such as breast, lung and kidney cancers. 8. Conclusion and perspectives In conclusion, recent and accumulating evidence suggests that AELs, such as edelfosine and ohmline, could constitute novel and lead compounds in raft-mediated cancer therapy. Indeed, edelfosine was found to reorganize lipid raft domains, leading to the formation of CASMER, a supramolecular structure that triggers tumor cell apoptosis, and ohmline allows Orai1–SK3 to move away from lipid rafts that reduces constitutive calcium entry, cancer cell migration and bone metastases (Fig. 5). It is well known that lipid rafts organize ion channels and their downstream acting molecules to modulate intracellular signaling, such as calcium signaling pathways regulating apoptosis and cell migration. Formation of such ion channel complexes, such as the SK3–Orai1 complex (Fig. 5), in lipid rafts represents a gain-of-function for tumor cell, a function which does not exist with the individual proteins or when the protein complex is not integrated into lipid rafts. An outcome of this feature is the opportunity given to therapists to address tumor development by altering the lipid composition of lipid rafts. On these grounds, further research on agents that reorganize lipid rafts, such as AELs, could be highly rewarding in the search for new cancer therapy approaches. This strategy is also worth being further investigated in view of the favorable toxicity profile of AELs, and their ability to promote apoptosis and inhibit metastasis in cancer, thus affecting two major hallmarks of cancer cells, such as apoptosis evasion and metastatic dissemination. Conflict of interest statement The authors declare no conflict of interest.

Please cite this article as: Jaffrès, P.-A., et al., Alkyl ether lipids, ion channels and lipid raft reorganization in cancer therapy, Pharmacology & Therapeutics (2016), http://dx.doi.org/10.1016/j.pharmthera.2016.06.003

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Acknowledgments This work was supported by the Agence Nationale de la Recherche No. ANR-08-EBIO-020-01, la Ligue Contre le Cancer, Region Centre (LIPIDS project of ARD2020-Biomédicaments), INSERM, CNRS, Cancéropôle Grand Ouest, the association “CANCEN”, Tours' Hospital oncology association “ACORT”, grants from Spanish Ministerio de Economia y Competitividad (SAF2011-30518, SAF2014-59716-R, and RD12/0036/0065 from Red Temática de Investigación Cooperativa en Cáncer, Instituto de Salud Carlos III, cofunded by the Fondo Europeo de Desarrollo Regional of the European Union), and European Community's Seventh Framework Programme FP7-2007–2013 (grant HEALTH-F2-2011-256986, PANACREAS). Ana Bouchet holds a post-doc mobility grant from PRESTIGEEuropean FP7/University of Tours. We thank Pr. S. Chevalier for its helpful comments regarding the role of lipids on the biology of tumor cells. We also thank Aurore Douaud-Lecaille and Isabelle Domingo for technical assistance and Catherine Leroy for secretarial support.

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