The antineoplastic properties of FTY720: evidence for the repurposing of fingolimod

Almost all drugs approved for use in humans possess potentially beneficial ‘off-target’ effects in addition to their principal activity. In some cases this has allowed for the relatively rapid repurposing of drugs for other indications. In this review we focus on the potential for re-purposing FTY720 (also known as fingolimod, Gilenya™), an immunomodulatory drug recently approved for the treatment of multiple sclerosis (MS). The therapeutic benefit of FTY720 in MS is largely attributed to the immunosuppressive effects that result from its modulation of sphingosine 1-phosphate receptor signalling. However, this drug has also been shown to inhibit other cancer-associated signal transduction pathways in part because of its structural similarity to sphingosine, and consequently shows efficacy as an anti-cancer agent both in vitro and in vivo. Here, we review the effects of FTY720 on signal transduction pathways and cancer-related cellular processes, and discuss its potential use as an anti-cancer drug.


Introduction
'On-target' effects; FTY720 modulation of sphingosine-1-phosphate signalling 'Off-target' effects of FTY720 -Sphingolipid metabolism -SET nuclear proto-oncogene/protein phosphatase 2A -Phosphatidylinositol-3 kinase (PI3K)/Akt -Other pathways Effect of FTY720 on the malignant phenotype -Cell death -Proliferation -Autophagy -Motility, invasion and metastasis -Epithelial to mesenchymal transition -Angiogenesis -Cancer-associated inflammation Second-Generation FTY720 Derivatives and Targeting Strategies -FTY720 derivatives that lack S1PR binding capability -FTY720 derivatives with enhanced SK inhibition -FTY720 with improved targeting Conclusions and future perspectives Introduction 2-Amino-2-[2-(4-octylphenyl)]-1,3-propanediol hydrochloride (FTY720 or fingolimod; commercially available as Gilenya TM ) is an immunosuppressive drug developed by the modification of myriocin (ISP-1), a metabolite of the fungus Isaria sinclairii [1,2] (Fig. 1). FTY720 was found to exert its immunosuppressive effects by mod-ulating sphingosine-1-phosphate (S1P) receptor signalling leading to sequestration of circulating lymphocytes in lymphoid tissues [3]. In 2010, FTY720 was approved by the FDA as a treatment for multiple sclerosis (MS) [2]. However, it has now become clear that FTY720 has a multitude of other effects on cells, many of which suggest it could be repurposed as an anti-cancer drug (Fig. 2). We now briefly review the impact of FTY720 on S1P signalling and other signal transduction pathways before considering its effects on cancer-related cellular processes.
'On-target' effects; FTY720 modulation of sphingosine-1-phosphate signalling Sphingosine-1-phosphate is a small bioactive lipid which exerts its effects following binding to one or more of at least five G protein coupled receptors, known as S1PR1-5. The consequences of S1P signalling are also partly determined by the relative levels of the different receptors on the cell surface. For example, S1PR1 couples to Gi to activate Ras/ERK and PI3-kinase/Akt pathways, leading to mitogenic and pro-survival signalling and cell migration [4]. In contrast, S1PR2 couples with multiple heterotrimeric G proteins, including G12/13 which exerts a potent inhibitory effect on Rac with consequent inhibition of cell migration [4]. S1P functions are also regulated in part by the balance between S1P and the death-promoting sphingolipids, ceramide and sphingosine [5,6]. Key regulators of this rheostat include: sphingosine kinase 1 (SPHK1) and SPHK2, which convert sphingosine to S1P; and several lipid phosphatases, including S1P phosphatase 1 and 2 (SGPP1 and SGPP2), which catalyse the conversion of S1P to sphingosine and S1P lyase, which irreversibly degrades S1P [5,6] (Fig. 3).
The classic mode of action of FTY720 is the binding of the drug to four of the S1PRs (S1PR1/3/4/5) after being phosphorylated (FTY720-P) principally by SPHK2 [7]. FTY720-P binds to the S1PRs at concentrations lower than 0.1 lM [8]. Although FTY720 has an initial agonist activity on the receptors, it subsequently causes their internalization thereby reducing receptor levels on the cell surface [9,10]. Because S1P-S1PR1 signalling is essential for T lymphocyte egress, FTY720 potently induces lymphocyte retention in peripheral lymphoid organs resulting in immunosuppression [11].
'Off-target' effects of FTY720 Apart from its classical 'on-target' action as a S1PR ligand, FTY720 also affects other signalling pathways when used at higher concentrations (greater than 2 lM) and we refer to these effects as 'off-target' actions of the drug. In the following section, we describe the main pathways that are affected by higher concentrations of FTY720.

Sphingolipid metabolism
In part. because of a structural similarity to sphingosine, FTY720 also influences other components of the sphingolipid pathway, [12]. FTY720 inhibits and reduces the expression of SPHK1 [10,[13][14][15]; as a sphingosine analogue, FTY720 is a competitive inhibitor of  SPHK1 and is also a non-competitive inhibitor of ATP binding to SPHK1 [16]. FTY720 has also been reported to inhibit and reduce the expression SPHK2 [17]. Further, FTY720 is a competitive inhibitor of ceramide synthase [18,19] and an inhibitor of S1P lyase [20]. Therefore, as a result of its multiple effects on sphingolipid metabolism, FTY720 leads to dysregulation of ceramide, sphingosine and S1P in vitro and in vivo [17, 21, 22].

SET nuclear proto-oncogene/protein phosphatase 2A
Protein phosphatase 2A (PP2A) is an enzyme with serine/threonine phosphatase activity that participates in a range of cellular mechanisms, including regulation of the cell cycle, apoptosis and cellular metabolism. Pathogenic mutations which result in decreased PP2A activity can lead to the development of colorectal and lung carcinomas and, therefore, PP2A is widely accepted as a tumour suppressor [23,24]. PP2A activity is inhibited following complex formation with the SET nuclear proto-oncogene [25]. FTY720 directly interferes with SET/ PP2A complexes and also reduces the expression of SET, both of which ultimately lead to the reactivation of PP2A [26,27]. Interestingly, although the interaction of FTY720 with SET/PP2A is independent of S1PRs, FTY720-P can also suppress PP2A activity via S1PR1 [28, 29].

Phosphatidylinositol-3 kinase/Akt
The phosphatidylinositol-3 kinase (PI3K)/Akt pathway participates in the regulation of cell metabolism, proliferation and survival, often via extensive crosstalk with other signalling pathways (including S1P and PP2A signalling) [30]. Upon activation, PI3K phosphorylates its substrate PIP2 to generate PIP3, which then activates Akt (v-akt murine thymoma viral oncogene homologue; protein kinase B). P13K signalling is disrupted in cancer following mutation of the PI3K gene itself or of other molecules that regulate its activity. One such molecule is phosphatase and tensin homologue deleted on chromosome 10 [14,33,34]. It is important to note that as the PI3K/Akt pathway can also be activated by S1P, it is likely that the inhibition of this pathway by FTY720 could occur via both S1P-dependent and -independent mechanisms.
Other pathways 14-3-3 proteins are a family of seven protein isoforms whose activities depend on the phosphorylation of serine/threonine residues. Once activated, these molecules bind with a diverse group of proteins that participate in signal transduction, which allows 14-3-3 proteins to regulate a wide range of regulatory processes, such as cell cycle [40] and apoptosis [41]. Similar to sphingosine, FTY720 directly modulates 14-3-3 proteins to facilitate their phosphorylation by protein kinase A (PKA) and possibly protein kinase C d [42], thereby influencing a vast array of cellular activities.
Reactive oxygen species (ROS) are generated as by-products of normal metabolism and are important regulators of cell signalling [43]. FTY720 has been shown to increase the permeabilization of lysosomal membranes and augment ROS release into the cytoplasm [21,44]. Other studies showed that FTY720 can increase ROS production [45][46][47] and this was found to be essential for the down-regulation of the anti-apoptotic protein, Mcl-1 in natural killer (NK) leukaemia cells [21], as well as for the activation of pro-apoptotic PKCd in hepatocellular carcinoma [34].
Protein phosphatase 2A activation appears to be essential in mediating the apoptosis induced by FTY720 in several haematological cancers, because inhibition of PP2A activity by okadaic acid rescued cell death induced by FTY720 [32,38,39]. ROS generation also contributes to apoptosis as FTY720 induced apoptosis can be partially rescued with a ROS scavenger [34, 45,68]. Furthermore, 14-3-3 phosphorylation was shown to be important in mediating FTY720induced apoptosis, because cell death was attenuated following transfection with a non-phosphorylatable 14-3-3zeta mutant [42]. FTY720 interactions with the S1PRs appear not to be involved in the apoptotic response because FTY720-P (which binds to S1PRs) did not kill a variety of cancer cell types that were sensitive to FTY720 [17, 50,59,69]. Moreover, pre-treatment of B-cell chronic lymphocytic leukemia (B-CLL) cells with S1P failed to alter the cytotoxic effects of FTY720 [38]. Nevertheless, the sphingolipid pathway may play a role in medi-ating the cytotoxic effects of FTY720 in some circumstances via the inhibition of SPHK1 by FTY720. For example, overexpression of SPHK1 rescued prostate cancer cells from FTY720-induced cell death, however, this effect was not observed in cells with silenced S1PRs [22]. These results suggest that SPHK1 inhibition, but not the interaction with S1PRs, may be important in mediating the cytotoxic effects of FTY720.
Necrotic cell death induced by FTY720 has also been observed. FTY720-treated ovarian and melanoma cells showed no evidence of caspase activation and the cells were not able to bind to Annexin V [48,62]. Similarly, death induced in the cell lines of neuroblastoma, acute lymphoblastic leukaemia cells, mantle cell lymphoma, other and B cell malignancies were also caspase-independent, although necrosis was not proven [17,38,46,47]. Further, in an interleukin (IL)-3 dependent murine haematopoietic cell line, FL5.12, in which apoptosis was disabled by overexpressing Bcl-2, FTY720 down-regulated nutrient transporter proteins which resulted in starvation-induced necrosis [59]. ROS production has also been identified as an important mechanism for FTY720-induced necrosis [46,47,62] FTY720 can also induce receptor interacting protein kinase 1-dependent necroptosis following the activation of PP2A [29]. Together, these findings demonstrate the ability of FTY720 to kill cancer cells by different mechanisms in a variety of cellular settings.
Some cancer cells that are resistant to conventional chemotherapy appear to be sensitive to FTY720. For example, FTY720 can kill imatinib-resistant gastrointestinal stromal tumour [70] and myeloid cells harbouring c-KIT mutations. Similarly, FTY720 was cytotoxic towards leukaemic cells that demonstrate resistance to tyrosine kinase inhibitors [33,39]. FTY720 could also kill ovarian cancer cells independent of their sensitivity towards cisplatin, paclitaxel or other chemotherapy [13,48]. Studies using FTY720 in combination with a variety of conventional chemotherapy agents have demonstrated additive or synergistic effects (Table 1). FTY720 has also shown a convincing ability to sensitize cancer cells to radiation; FTY720 reduced the activation of Akt and down-regulated survivin, both of which were induced by radiation and were implicated in the radio-resistance of a breast cancer cell line [71]. In addition, FTY720 increased the radio-sensitivity of prostate cancer cells overexpressing miR-95, a microRNA associated with resistance to radiation) [72]. Similarly, the combination of FTY720 and radiation showed enhanced SK1 inhibition and tumour suppression in a mouse xenograft model of prostate cancer [22].

Autophagy
Autophagy is a physiological process in which damaged organelles form an autophagosome which is subsequently digested by lysosomal enzymes. The resulting metabolites are either recycled or used as a short-term energy supply in times of cellular stress. Autophagy plays an ambiguous role in cancer progression, as it can induce prolonged survival of cancer cells by conserving energy or lead to cell death [74]. FTY720 can increase the accumulation of autophagosomes in many malignancies either by inducing autophagosome formation [46,48,59,68] or by blocking the fusion of autophagosomes and lysosomes (autophagic flux) [44]. FTY720-induced autophagy was found to be protective against the cytotoxic nature of FTY720, which was demonstrated by the ability of 3-methyladenine, an autophagy inhibitor, to enhance the cell death induced by FTY720 [46,48,62]. In addition, autophagy-deficient murine embryonic fibroblasts were more sensitive to FTY720-induced cytotoxicity [59]. Interestingly, FTY720-P was also found to induce autophagy [46], implying the involvement of S1P and the S1PRs. In acute lymphoblastic leukaemia cells, FTY720 was shown to mediate autophagy by down-regulating Mcl-1, which inhibits Beclin-1 [46], an important inducer of autophagy [74]. Similarly, Beclin-1 was up-regulated by FTY720 in ovarian cancer cells [48]. By contrast, FTY720-induced autophagy promoted apoptosis in multiple myeloma cells in which both autophagy and apoptosis were mediated through ROS generation, an effect that was attributed to the degradation of anti-apoptotic protein, Mcl-1 and survivin [68].

Motility, invasion and metastasis
At concentrations below those that cause cytotoxicity, FTY720 treatment decreased the migration and invasive ability of glioblastoma and prostate cancer cells in in vitro assays [53,75,76]. The anti-migratory and/or anti-invasive effects of FTY720 have also been reported in other cancer cell lines, such as those from ovarian cancer [13], hepatocellular carcinoma [77][78][79], pancreatic cancer [80] and cholangiocarcinoma [65]. These results are supported by observations that FTY720 induced cytoskeletal disorganisation in prostate cancer cells [53] and also decreased and deformed microfilaments, filopodia and microvilli on the cell surface of murine breast cancer cell lines [52]. In addition, FTY720 has been shown to suppress lymph node and organ metastasis in many in vivo cancer models [45,52,65,69,79,81], indicating that FTY720 might be effective in managing late stage disease.
The concentration of FTY720 needed for the drug to inhibit tumour cell migration/invasion is lower than that required to induce cytotoxicity in both in vitro (2 lM or less) [13,53,61,75,78] and in vivo studies (2 mg/kg) [52]. This suggests, therefore, that the SPHK1/S1P/S1PR signalling pathway is important in mediating the effects of FTY720 on migration/invasion and metastasis. FTY720-P inhibited S1P-induced migration of classical Hodgkin lymphoma cells by modulating S1PR1 [82] and SK1 inhibition with FTY720 reduced the migration of ovarian cancer cells [13]. FTY720 affects a number of pathways that are known to be downstream of the S1PRs, such as the Rho family of small GTPases, which are important regulators of cell mobility [83]. FTY720 down-regulated the active form of RhoA in pancreatic cancer cells [53], reduced levels of active Rac in hepatocellular carcinoma [78,79] as well as decreasing levels of ROBO1 and ROCK1 (targets of RhoA) in glioblastoma cells. FTY720 also decreased the expression of metalloproteinases (MMP-2 and MMP-9) and increased tissue inhibitors of metalloproteinases (TIMP-1 and TIMP-2) [13,75]. The PI3K/Akt pathway has been implicated in FTY720-induced motility [75,78], although this could also be the downstream of S1PRs.

Epithelial to mesenchymal transition
Epithelial to mesenchymal transition (EMT) is a process by which epithelial cells undergo molecular and morphologic changes to resemble the mesenchymal phenotype, which leads to the acquisition of migratory and invasive capacity, evasion of apoptosis and senescence, as well as ability to resist chemotherapy [84]. The growth of xenografts derived from cisplatin resistant lung cancer cells showing features of EMT was suppressed by FTY720, both alone or in combination with cisplatin [85]. These effects were attributed to the ability of FTY720 to modulate the PP2A/SET interaction together with a concomitant increase in E-cadherin and the Snail transcription factor, as well as a decrease in vimentin expression [85]. Similar observations were made in cholangiocarcinoma [65] and glioblastoma [75], where FTY720-treated cells showed higher expression of E-cadherin and reduced expression of N-cadherin, vimentin and Twist1 [65,75]. In  androgen-independent prostate cancer cells, Runx2 modulates EMT by switching of E-cadherin to N-cadherin and FTY720 down-regulated Runx2 thereby reversing the cadherin switch [76].

Angiogenesis
Angiogenesis is the process by which new blood vessels are formed to sustain nutrient and oxygen requirements of actively proliferating cells and is important for the sustained growth of most tumours [86]. FTY720 has been reported to inhibit angiogenesis in several xenograft cancer models [61,65,79,81]. Similarly, FTY720 attenuated both S1P-and VEGF-driven angiogenesis in an agar chamber model in vivo [81] and a Matrigel plug in vivo assay for Lewis lung carcinoma [87]. Furthermore, FTY720 normalized the vasculature within mammary tumours in rats [88] and abrogated increased vascular permeability [61,81], both of which can promote the cytotoxic effects of chemotherapy and radiotherapy [89,90]. In addition, low doses of FTY720 did not kill B16/BL6 melanoma cells in vitro but reduced the growth of these cells and inhibited neovascularization in vivo, suggesting the indirect killing of tumour cells by reducing tumour vascularity [81]. FTY720 has been shown to inhibit angiogenesis by a number of mechanisms. For example, FTY720 was found to reduce the migration of human umbilical vein endothelial cells (HUVEC) [61,63,81,87] and to block the recruitment of vascular smooth muscle cells (VSMC) by S1P, endothelial cells or tumour cells [88]. S1PR antagonizm by FTY720 is important in mediating these anti-angiogenic effects as they are induced at low doses [61,81,87] and by FTY720-P [81]. In support of these observations, FTY720 reversed the effect of S1P on VSMC/HUVEC migration and the formation of blood vessels by a mechanism involving S1PR1/3 [81,88,91]. In addition, FTY720 down-regulates VEGF, an important angiogenic inducer [63,92] as well as reduces the expression of chemokines, i.e. CXCL10, CXCR3 and CXCR4 [92].

Cancer-associated inflammation
It is now recognized that inflammation can promote tumourigenesis [93]. FTY720 suppressed azoxymethane-induced colonic inflammation in mice and suppressed the subsequent development of tumours by down-regulating SPHK1 and S1PR1, which is important for persistent NF-jB and STAT3 activation, as well as IL-6 production in this model [10]. In addition, FTY720 has been reported to down-regulate the pro-inflammatory mediators CXCL10, VEGF, CXCR4 and CXCR3 and reduce hepatic ischaemia-reperfusion injury, which otherwise contributes to metastasis in rats with hepatic tumours [92].
Second-generation FTY720 derivatives and targeting strategies FTY720 derivatives that lack S1PR binding capability As many of the anti-cancer effects of FTY720 are independent of S1PRs and there are possible side effects associated with antagonizing S1P signalling, a non-immunosuppressive FTY720 analogue, OSU-2S was developed that cannot be phosphorylated by SPHK2 and does not induce S1PR1 internalization [98]. Compared to FTY720, OSU-2S demonstrated more cytotoxicity and selectivity (in relation to normal liver cells) in hepatocellular carcinoma, both in vitro and in vivo, [98]. OSU-2S was also shown to induce   [99]. Two other FTY720 derivatives, (S)-FTY720-OMe, (S)-FTY720-regioisomer, were found to reduce survival of chronic myeloid leukemia (CML) haematopoietic stem cells (HSC) but not normal HSCs [58] and caused PP2A activation without stimulating S1PR1 internalization and B cell lymphopenia [58]. Both OSU-2S and (S)-FTY720-regioisomer were also found to be more potent than FTY720 in reducing the clonogenic survival of Jak2-driven haematological malignancies [28]. Another FTY720 analogue devoid of the ability to be phosphorylated is AAL-149 [59]. This drug demonstrated the same potency and mechanism of selective cytoxicity as FTY720 in patient-derived leukaemic cells [59]. In 2013, Fransson and colleagues designed novel stereochemically constrained analogues of FTY720 and showed that one of these analogues had an enhanced anti-leukaemic activity compared to FTY720 [100]. However, the same enhanced potency was not observed for prostate cancer cells [100].

FTY720 derivatives with enhanced sphingosine kinase inhibition
Efforts have also been made to chemically modify FTY720 to improve its efficacy as a SPHK inhibitor. This has resulted in the generation of two compounds; (S)-FTY720 vinylphosphonate and (R)-FTY720 methyl ether (ROME). (S)-FTY720 vinylphosphonate inhibits and reduces the expression of SPHK1 [15,16,101,102], resulting in apoptosis of prostate cancer cells [101] and human pulmonary smooth muscle cells [15]. In addition to inhibiting SPHK1, (S)-FTY720 vinylphosphonate abrogated the S1P-stimulated rearrangement of actin in breast cancer cells [103]. ROME, on the other hand, is a derivative which selectively inhibits and down-regulates SPHK2 [104,105] in turn inhibiting DNA synthesis and preventing S1P-mediated rearrangement of actin in MCF-7 cells [105]. However, ROME did not induce apoptosis in androgen-sensitive LNCaP prostate cancer cells [106].

FTY720 with improved targeting
To reduce unwanted toxicity, recent studies have also examined the feasibility of improved targeting of FTY720. Liposomal formulation of FTY720 improved the stability of FTY720 in aqueous buffer without affecting the cytotoxicity of CLL cells [107]. When this formulation was coupled to an antibody (i.e. CD19, CD20 and CD37), a superior specificity against CLL cells was observed [107]. Similarly, liposomal-antibody packaging of OSU-2S allowed this drug to selectively target CLL cells, sparing normal B cells [99]. Dual antibody immunoliposomes have been developed as vehicles for targeted delivery [108], which resulted in enhanced delivery of FTY720 and increased apoptosis in CLL cells compared to the single antibody liposomal targeting [108].

Conclusions and future perspectives
The ability of FTY720 to target multiple signalling pathways which control cell proliferation, death, motility, angiogenesis and inflammation (Fig. 4), suggests that this drug is not only likely to be useful against a wide range of tumours containing different molecular abnormalities, but also that it could reduce the likelihood of resistance resulting from the activation of other compensatory pathways [94]; using a single drug that targets multiple pathways would seem to be an attractive alternative to the use of combinations of drugs with narrower specificity to reduce the likelihood of developing resistant disease [94]. The toxicity profile of FTY720 is well described in MS patients and includes immunosuppression, bradycardia and increased risk of melanoma [95][96][97]. However, it is difficult to predict the toxicity associated with the use of FTY720 in cancer patients, because dose and duration of treatment may be different; there is already evidence from in vitro studies that the dose required to achieve an anticancer effect is higher than that necessary to antagonize S1PR signalling. The long-term adverse effects of FTY720 treatment are still to be fully determined, but will obviously be important considerations for the potential future use of this drug in cancer patients.
By targeting a range of processes implicated in tumourigenesis, FTY720 is a promising anticancer agent across a broad range of malignancies that has the potential and meets a number of accepted criteria for drug repurposing [109] (Fig. 2). The secondgeneration derivatives of FTY720 have higher efficacy, lower toxicity and better selectivity (Table 2). However, as the precise effects of FTY720 on molecular signalling pathways and clinical phenotypes appear to be cell-type dependent, further studies are required to fully evaluate the utility of FTY720 and its derivatives in different cancer settings.