Cancer cells are hypersensitive to nutrient limitation because oncogenes constitutively drive glycolytic and TCA (tricarboxylic acid) cycle intermediates into biosynthetic pathways. As the anaplerotic reactions that replace these intermediates are fueled by imported nutrients, the cancer cell's ability to generate ATP becomes compromised under nutrient-limiting conditions. In addition, most cancer cells have defects in autophagy, the catabolic process that provides nutrients from internal sources when external nutrients are unavailable. Normal cells, in contrast, can adapt to the nutrient stress that kills cancer cells by becoming quiescent and catabolic. In the present study we show that FTY720, a water-soluble sphingolipid drug that is effective in many animal cancer models, selectively starves cancer cells to death by down-regulating nutrient transporter proteins. Consistent with a bioenergetic mechanism of action, FTY720 induced homoeostatic autophagy. Cells were protected from FTY720 by cell-permeant nutrients or by reducing nutrient demand, but blocking apoptosis was ineffective. Importantly, AAL-149, a FTY720 analogue that lacks FTY720's dose-limiting toxicity, also triggered transporter loss and killed patient-derived leukaemias while sparing cells isolated from normal donors. As they target the metabolic profile of cancer cells rather than specific oncogenic mutations, FTY720 analogues such as AAL-149 should be effective against many different tumour types, particularly in combination with drugs that inhibit autophagy.
- nutrient limitation
Although it was recognized almost a century ago that cancer cells are highly glycolytic, it has only recently become appreciated that the enhanced rate of glycolysis in tumour cells most likely reflects a need for accelerated biosynthesis. All rapidly proliferating cells use glycolytic and TCA (tricarboxylic acid) cycle intermediates as building blocks for nucleotide, membrane and protein synthesis [1,2]. The intermediates consumed in these biosynthetic reactions are replenished by anaplerotic reactions that depend upon imported nutrients. All rapidly proliferating cells rely on these processes to support biosynthesis. However, the regulation of anabolic metabolism is different in normal and transformed cells. In normal cells, anabolism is driven by growth factors and is sensitive to extracellular nutrient levels. When nutrients become limiting, normal cells make adaptive changes in their metabolism, undergoing cell cycle arrest and becoming quiescent and catabolic. Cancer cells, in contrast, continue biosynthesis despite nutrient deprivation, because anabolism is driven by constitutively active oncogenes and uncoupled from environmental cues by the deletion or inactivation of negative regulators of growth. As glycolytic and TCA cycle intermediates continue to be utilized but cannot be replenished, ATP generation is eventually compromised in nutrient-restricted cancer cells [3,4]. In addition to the problems associated with constitutive anabolism, cancer cells also have defects in autophagy, the catabolic process through which cells derive nutrients from self-digestion . Together, constitutive anabolism and defective autophagy trigger a bioenergetic crisis in transformed cells under conditions that produce proliferative arrest and quiescence in normal cells. Consistent with this model, hyperactivation of growth-promoting oncogenes such as the serine/threonine kinase Akt, the GTPase K-Ras and the transcription factor Myc sensitizes cells to nutrient limitation [4,6–8]. Inactivation of the tumour-suppressor proteins that orchestrate quiescence and catabolism during nutrient stress also increases dependence on extracellular nutrients. For example, loss of the tuberous sclerosis complex that limits the activity of the mTOR (mammalian target of rapamycin) kinase, the AMPK (AMP-activated protein kinase) that co-ordinates the cellular response to energy stress, the serine/threonine kinase LKB1 that regulates AMPK and related kinases or the transcription factor p53 sensitizes cells to starvation [9–13]. Because many different mutations that transform cells increase dependence on imported nutrients, limiting access to nutrients could be a means to selectively kill diverse types of cancer cells.
Some currently available cancer therapies work by limiting nutrient availability. Angiogenesis inhibitors restrict nutrient delivery to expanding tumours by limiting the growth of new blood vessels. However, these drugs have several significant drawbacks: (i) they select for resistant cancer cells that are more aggressive and invasive [14,15], (ii) leukaemias are unlikely to be nutrient-limited by this approach as they reside in the bloodstream, and (iii) as small tumours can obtain nutrients by diffusion, blocking angiogenesis would not eliminate all cancer cells. L-asparaginase, an enzyme that degrades the amino acid asparagine, is used to treat acute lymphocytic leukaemia . Leukaemia cells cannot synthesize sufficient quantities of asparagine to meet their high demand, whereas asparagine production can counterbalance the effect of L-asparaginase in normal cells. PEGylated arginine deiminase is effective in animal studies, but against tumour cells with insufficient capacity to synthesize arginine . Although beneficial, these enzymes target individual nutrients and are only active against a limited spectrum of tumours. If therapies that restrict cellular access to multiple nutrients can be developed, they should be more broadly effective and might limit the ability of tumour cells to develop resistance by switching to alternative fuels. If these compounds block nutrient uptake rather than nutrient delivery, many of the limitations of angiogenesis inhibition could be avoided.
Our previous work demonstrated that multiple mammalian nutrient transporter proteins could be targeted for down-regulation by the sphingolipid ceramide . Because ceramide is extremely hydrophobic and readily metabolized, we investigated whether sphingolipid-based drugs with superior pharmacological properties might selectively kill cancer cells through a similar mechanism. The water-soluble sphingosine analogue FTY720 is a highly effective non-toxic anti-cancer agent in a wide variety of animal model systems. FTY720 limits the growth of the primary tumour and metastatic nodules in breast cancer and hepatocellular carcinoma models [19,20], inhibits the growth of bladder cancer and androgen-independent prostate cancer xenografts [21,22], and protects mice from BCR (breakpoint cluster region)-ABL-driven leukaemia . FTY720 is selectively toxic to transformed cells, and mice can be maintained on an antineoplastic dose for as long as 26 weeks with no ill effects [22–24]. Although FTY720 is effective in animal cancer models, it cannot be used in human cancer patients, because it causes a significant reduction in heart rate at the antineoplastic dose by activating S1P (sphingosine 1-phosphate) receptors. As the mechanism behind FTY720's anti-cancer activity was not understood and it was unclear whether the antineoplastic and toxic effects of FTY720 were separable, FTY720 derivatives have not been pursued as potential cancer therapies.
In the present study we report that FTY720 selectively kills cancer cells by down-regulating nutrient transporter proteins. Furthermore, we demonstrate that an FTY720 analogue that lacks FTY720's dose-limiting toxicity retains its ability to trigger nutrient transporter loss and selectively kill cancer cells. These studies suggest that FTY720 analogues could be a novel, safe and effective means to starve constitutively anabolic cancer cells to death in the midst of abundant extracellular nutrients.
Chemicals were from Cayman, EMD, Biomol or Sigma. Antibodies against the following were used: murine 4F2hc (4F2 heavy chain), human CD4, B220 (CD45.2) from eBioscience; murine transferrin receptor and human 4F2hc from BD Biosciences; human GLUT1 (glucose transporter 1) from Novus; murine GLUT1 from RDI (however, recent lots of antibody did not recognize mouse GLUT1); LC3 from MBL; and others from Cell Signaling Technology. The Bcl-2 (B-cell lymphoma 2) G154A plasmid was supplied by Stanley Korsmeyer via Addgene (#8751). The GFP (green fluorescent protein)–HTLV-2-RBD construct was provided by Marc Sitbon (Institut de Génétique Moléculaire de Montpellier, Montpellier, France). Phosphorylated FTY720 and AAL-149 were supplied by Volker Brinkmann (Novartis Institutes for Biomedical Research, Basel, Switzerland).
The IL-3 (interleukin 3)-dependent murine haemopoetic cell line FL5.12 was maintained at 250000–700000 cells/ml in RPMI 1640 medium (Mediatech) supplemented with 10% FCS (fetal calf serum) (Sigma–Aldrich), 500 pM recombinant mouse IL-3 (eBioscience), 10 mM Hepes (Mediatech), 55 μM 2-mercaptoethanol (Sigma–Aldrich), antibiotics and L-glutamine (Mediatech). RPMI lacking glucose and amino acids was made from chemical components. For nutrient deprivation, cells were maintained in this medium supplemented with 10% dialysed FCS (Invitrogen). To adapt cells to low nutrient conditions, amino acid and glucose-free medium was supplemented with standard FCS. Oleic acid was conjugated to fatty acid-free BSA at a molar ratio of 3:1. AMPK−/− MEFs (murine embryonic fibroblasts) lacking both α subunits, originally developed by Keith Laderoute, were provided by Reuben Shaw (Molecular and Cell Biology Laboratory, Salk Institute, La Jolla, U.S.A.). Atg5−/− MEFs were provided by Noboru Mizushima (Department of Physiology and Cell Biology, Tokyo Medical and Dental University, Tokyo, Japan). Rab7+/− MEFs were generated from mice conditionally deficient in Rab7 developed in the Edinger laboratory. IL-3-dependent Bax−/− (Bcl-2-associated X protein) Bak−/− (Bcl-2 homologous antagonist/killer) cells were provided by Tullia Lindsten (Cancer Biology and Genetics, Memorial Sloan Kettering Cancer Centre, New York, U.S.A.). Where Fumonisin B1 was used, cells were pre-treated for 1 h with twice the final concentration. Cells were pretreated for 4 h with okadaic acid. Cells were spun down and resuspended in fresh medium daily in experiments that lasted longer than 72 h. Methocult colony assays were performed as described previously . Peripheral blood mononuclear cells isolated from three normal volunteers were plated in methylcellulose medium containing recombinant human IL-3 (30 ng/ml) and GM-CSF (granulocyte/macrophage colony-stimulating factor; 500 ng/ml) at 1×106 cells/ml and granulocyte/macrophage colonies were counted at 15 days. Research with patient-derived samples was carried out in accordance with the Declaration of Helsinki (2000) of the World Medical Association and was approved by the Institutional Review Board for Human Use of Loma Linda University. Consent was obtained from each patient after full explanation of the purpose, nature and risk of all procedures used.
Amino acid uptake
FL5.12 cells were suspended at low density in fresh warm complete RPMI in the presence or absence of 5 μM FTY720. After 2 h, 1 μCi/ml [3H]arginine (MP Biomedical) was added and cells were incubated at 37°C, or on ice where indicated, for 3 h. To stop uptake, RPMI containing 100×unlabelled arginine was added on ice and cells were washed twice with ice-cold RPMI containing 10×arginine (2 mg/ml) before lysis. [3H]Arginine in cell lysates was measured by scintillation counting. Uptake was linear over the course of the experiment (R2=0.99).
Flow cytometry assays and microscopy
Analysis was restricted to viable cells; cells that excluded vital dyes [propidium iodide or DAPI (4′,6-diamidino-2-phenylindole)] were deemed viable. Surface GLUT1 was measured using GFP–HTLV-2 RBD as described previously . Cell accumulation was measured by flow cytometry by collecting for a fixed interval, generally 30–60 s. For immunofluorescence, cells were fixed with 1% paraformaldehyde and permeabilized with 0.3% saponin. For electron microscopy, cells were fixed in 2.5% glutaraldehyde/2.5% formaldehyde in 0.1 M sodium cacodylate buffer (Electron Microscopy Systems) and stored at 4°C until embedding. Cells were post-fixed in 1% OsO4 on ice for 1 h and contrasted with 1% uranyl acetate at room temperature (25°C) for 1 h, dehydrated in ethanol, and embedded in Epon 812. Ultrathin sections (~60 nm) were cut on a Reichert Ultracut Ultramicrotome. Sections were stained again in 2% uranyl acetate for 2 min, followed by Reynold's lead citrate for 2 min. Sections were examined on a Philips CM10 transmission electron microscope and micrographs were taken with a Gatan Ultrascan US1000 digital camera.
Ceramide levels in Bax−/−Bak−/− cells were measured by LC (liquid chromatography) MS performed in the Lipidomics Core Facility at the Medical University of South Carolina. For experiments using FL5.12 cells, flow injection ESI-MS/MS (electrospray ionization tandem MS) analysis was performed using a Waters Quattro Ultima electrospray tandem mass spectrometer. Detailed MS methods for the experiments using FL5.12 cells are provided in the Supplementary Experimental section (at http://www.BiochemJ.org/bj/439/bj4390299add.htm).
Sub-lethally irradiated (4.5 Gy) 6–9-week old female Balb/c mice were injected with 500000 BCR-ABL p190-expressing FL5.12 cells and leukaemia was allowed to progress for 6–7 days (FL5.12 cells were originally derived from Balb/c mice ). Mice were then treated with FTY720 (10 mg/kg of body weight intraperitoneally daily) or with vehicle (water) alone for 3 days, at which point splenocytes were harvested and analysed. A two-tailed t test was used to determine statistical significance. All animal protocols were approved by the Institutional Animal Care and Use Committee of University of California, Irvine.
FTY720 triggers nutrient transporter loss in mammalian cells
FTY720 is structurally similar to ceramide, a sphingolipid that we have previously shown produces nutrient transporter down-regulation . Studies in yeast suggested that FTY720 might also limit nutrient permease expression . We used FL5.12 murine haemopoetic cells to test whether FTY720 could induce transporter loss in mammalian cells because this cell line expresses high levels of nutrient transporters and is sensitive to ceramide-induced transporter loss . In addition, FL5.12 cells are particularly useful for studies assessing the contribution of the cellular metabolic state to drug toxicity because they are immortalized but not transformed. FL5.12 cells are immortalized and will undergo unlimited proliferation in culture as long as they are supplied with IL-3. However, their rapid growth is driven by growth factors rather than oncogenes, and thus their anabolic rate can be reduced by gradually decreasing growth factor or nutrient levels. Thus the response of genetically identical cells with different metabolic profiles can be compared. We first evaluated the effect of FTY720 on the 4F2hc, a type II membrane protein that associates with several different light chains to form heterodimeric amino acid transporter complexes [29,30]. FTY720 rapidly decreased 4F2hc surface levels with kinetics similar to those previously described for ceramide (Figure 1A, ). The B-cell marker protein B220 was, in contrast, not down-regulated (Figure 1B). The transferrin receptor, which traffics via a distinct pathway from 4F2hc , was also minimally affected (Figure 1C). Similarly to the 4F2 complex, the glucose transporter GLUT1 and CAT-1 (cationic amino acid transporter 1) are found in lipid raft domains [31,32]. These nutrient transporters were also down-regulated by FTY720 (Figure 1D). As expected from these results, amino acid uptake was markedly reduced by FTY720 (Figure 1E). FTY720 inhibits proliferation and triggers cell death in a wide variety of cancer cell types [19–24]. In keeping with these findings, FTY720 reduced surface 4F2hc and GLUT1 levels in HeLa cervical carcinoma cells, DU145 and PC3 prostate cancer cells, and in Sup-B15 human leukaemia cells (Figures 1F–1H). Thus FTY720 co-ordinately down-regulated nutrient transporter proteins in diverse mammalian cell types.
FTY720 induces starvation despite abundant extracellular nutrients
Given that FTY720 produced profound nutrient transporter loss (Figure 1), we hypothesized that FTY720 kills cells by restricting access to extracellular nutrients. The dose of FTY720 necessary for nutrient transporter loss and cell death was tightly correlated (Figures 2A and 2B). Reduction of 4F2hc levels by >30% resulted in the death of all of the cells in the culture. When transporter levels remained above 70% of control levels, viability was minimally affected but proliferation was reduced (Figures 2B and 2C). Similar effects were observed in HeLa, DU145, PC3 and Sup-B15 cells (results not shown). To determine whether transporter loss was sufficient to induce bioenergetic stress, we assessed whether FTY720 induced autophagy. Through autophagy, nutrient-limited cells encapsulate and degrade non-essential components to recycle intracellular nutrients . Consistent with profound transporter loss, LC3-II, an autophagosome marker, accumulated to an equivalent degree following FTY720 treatment and extracellular nutrient limitation (Figure 2D). An increase in autophagosome number was also observed in FTY720-treated cells (Figure 2E and Supplementary Figure S1A at http://www.BiochemJ.org/bj/439/bj4390299add.htm). Autophagic flux was increased by FTY720 treatment as LC3-II accumulation was enhanced by chloroquine addition (Figure 2D). These results are consistent with autophagy induction in response to intracellular nutrient limitation.
To confirm that FTY720-induced autophagy was a homoeostatic response to starvation, we assessed the consequences of limiting autophagy. Atg5−/− MEFs that are unable to form autophagosomes were hypersensitive to FTY720-induced cell death (Figure 2F), demonstrating that autophagy is a protective response. Autophagy generates nutrients but also promotes cellular health by removing damaged organelles that consume ATP and generate free radicals. Encapsulating these organelles in autophagosomes may limit their access to substrates, sparing nutrients and relieving toxicity. If, however, the protective function of autophagy in FTY720-treated cells is nutrient generation, autophagosome degradation would be required. Rab7 is necessary for the degradation of autophagosomes in the lysosome but not for their formation . Consistent with a model where autophagy is required to generate nutrients, Rab7+/− MEFs were hypersensitive to FTY720 (see Supplementary Figure S1B). AMPK co-ordinates the cellular response to energy stress, increasing autophagy and inhibiting biosynthesis . AMPK-deficient MEFs were also hypersensitive to FTY720 (Figure 2G). mTOR is also a nutrient sensor, exhibiting reduced activity when amino acids are limiting . Phosphorylation of the downstream mTOR target, ribosomal protein S6, was also decreased by FTY720 (Figure 2H). In summary, FTY720-induced nutrient transporter loss is sufficient to induce starvation in the midst of abundant extracellular nutrients.
FTY720 kills cells by down-regulating nutrient transporter proteins
If FTY720 kills cells by causing nutrient transporter loss, membrane-permeant nutrients that do not require transporters to enter cells should be protective. Two cell-permeant nutrients, dimethyl succinate and oleic acid, limited FTY720-induced death without blocking nutrient transporter loss (Figure 3A and Supplementary Figure S2A at http://www.BiochemJ.org/bj/439/bj4390299add.htm). Interfering with transporter down-regulation should also rescue cells from FTY720. FTY720 affects nutrient transporter proteins that are internalized via a clathrin-independent, lipid raft-dependent pathway [31,32,37]. Consistent with this, the lipid raft-disrupting agent nystatin increased basal levels of surface 4F2hc and maintained 4F2hc levels in the presence of FTY720 (Figure 3B). Although nystatin is itself somewhat toxic, increased transporter levels led to a parallel increase in cell viability (Figure 3C). If transporters are limiting, FTY720-treated cells should be hypersensitive to extracellular nutrient limitation. In fact, under conditions where neither nutrient limitation nor FTY720 were toxic, striking synergy was observed when these treatments were combined (Figure 3D). Together, these results support a model where FTY720 kills cells by triggering transporter down-regulation.
Apoptosis will ensue if nutrient stress is prolonged or severe. However, bioenergetic stress will eventually lead to necrotic death if apoptosis is disabled [18,38]. To determine whether FTY720-induced transporter loss led to sufficient bioenergetic stress to kill apoptosis-deficient cells, we used cells overexpressing Bcl-2 G145A, a mutant form of Bcl-2 that blocks apoptosis but does not interfere with autophagy . Bcl-2 G145A expression abrogated cell death following the loss of growth factor signalling, a purely apoptotic stimulus (Supplementary Figure S2B). However, Bcl-2 G145A overexpression did not prevent FTY720-induced death (Figures 3E and 3F). FTY720 did not induce the DAPI-negative, Annexin V-positive population characteristic of apoptosis that is observed following growth factor withdrawal (Figure 3E). The nuclear condensation and fragmentation that is characteristic of apoptotic death was also not observed (Figure 3F). Furthermore, FTY720 rapidly killed Bax−/−Bak−/− cells that are highly resistant to apoptotic forms of death such as growth factor withdrawal and staurosporine treatment (Figure 3G) . From these experiments, we conclude that FTY720 induces bioenergetic stress severe enough to trigger necrosis when apoptosis is blocked.
Bioenergetic state determines sensitivity to FTY720-induced transporter loss
If FTY720 kills cells by limiting nutrient uptake, drug sensitivity should parallel bioenergetic demand. To test this hypothesis, we generated genetically identical cells with different metabolic programmes. As rapid growth is not oncogene-driven in FL5.12 cells, their bioenergetic programme is flexible and can be altered by varying the culture conditions . By gradually reducing the nutrients in the culture medium, we produced FL5.12 cells able to grow in only 5% of the normal level of amino acids and glucose. These cells exhibited stress response hormesis, a phenomenon where sub-lethal exposure to a stress triggers a protective response that confers resistance to a lethal level of the same form of stress . FL5.12 cells adapted to low nutrient medium were highly resistant to FTY720-induced death (Figure 4A), demonstrating that FTY720 and extracellular nutrient limitation induce similar forms of stress. As an alternative means to reduce metabolic demand, FL5.12 cells were adapted to grow in low levels of IL-3. Although acute IL-3 withdrawal triggers apoptosis in FL5.12 cells (Figures 3E and 3F), gradually reducing IL-3 concentration slows anabolism, reducing glycolysis and increasing cellular dependence on oxidative phosphorylation [41,43]. FL5.12 cells adapted to low levels of IL-3 were resistant to FTY720-induced cell death (Figure 4B), consistent with a model where a high rate of anabolism sensitizes cells to the drug. This interpretation is supported by the fact that IL-3 is a critical survival factor in this cell line, yet reducing its concentration in the medium was protective. Anabolism was also reduced pharmacologically. The mTOR complex 1 inhibitor rapamycin reduces glycolysis and protein synthesis [36,44]. Consistent with its ability to rescue cells from starvation [9,10,13], rapamycin afforded substantial protection from FTY720-induced cell death (Figure 4C). Weaning cells off glycolysis in advance of exposure to FTY720 by pretreating with a low dose of the glycolysis inhibitor 2-DG (2-deoxy-D-glucose) was also protective (Figure 4D); as expected, pre-treating with D-glucose was not beneficial.
Lipid rafts are signalling platforms, and it was possible that disruption of growth factor receptor signalling contributed to the toxicity of FTY720. However, IL-3-dependent phosphorylation of STAT5 (signal transducer and activator of transcription 5) and ERK1/2 (extracellular-signal-regulated kinase 1/2) was not disrupted by FTY720, and Akt phosphorylation was only marginally affected (Figures 4E and 4F). Moreover, slowing anabolism with rapamycin or 2-DG did not protect cells from the loss of growth factor receptor-dependent signal transduction (Supplementary Figures S3A and S3B at http://www.BiochemJ.org/bj/439/bj4390299add.htm). Taken together, these experiments show that FTY720 induces death secondary to starvation rather than by compromising growth factor receptor-dependent signalling.
The anti-neoplastic dose of FTY720 triggers nutrient transporter loss in vivo
To test whether FTY720-induced nutrient transporter down-regulation might explain its activity in cancer models, we utilized a BCR-ABL-driven leukaemogenesis model in which FTY720 is effective . This model system was selected because leukaemia cells can be recovered and analysed by flow cytometry with minimal processing. To track the leukaemic cells, BCR-ABL p190 expression was coupled to human CD4 expression via an internal ribosome entry site. BCR-ABL p190 expression rendered FL5.12 cells IL-3-independent as expected, but did not block FTY720-induced transporter loss and death in vitro (see Supplementary Figures S4A and S4B at http://www.BiochemJ.org/bj/439/bj4390299add.htm). In vivo, FTY720 significantly decreased the average leukaemic burden in the spleen from 40% to 18% (Figure 5A); mice were treated for only 4 days to ensure that sufficient numbers of leukaemic cells would be recovered to allow further analysis. Importantly, FTY720 induced nutrient transporter down-regulation in vivo (Figure 5B). This decrease was detected both prior to and following normalization to B220, a molecule that is not down-regulated by FTY720 (Supplementary Figure S4B). FTY720 treatment led to reduced S6 phosphorylation (Figure 5C), suggesting that transporter loss induced nutrient stress in vivo. Finally, FTY720 selectively killed leukaemic cells in vivo (Figure 5D). In summary, the established anti-neoplastic dosing schedule for FTY720 produces tissue concentrations of the drug that are sufficient to trigger nutrient transporter loss.
FTY720-induced nutrient transporter loss does not require increased ceramide production
Ceramide down-regulates nutrient transporter proteins . FTY720 inhibits both S1P lyase  and sphingosine kinase 1 , potentially increasing ceramide production from sphingosine via the salvage pathway (Figure 6A). We therefore hypothesized that FTY720 triggers nutrient transporter loss by increasing ceramide generation. Consistent with a previously published study , we found that FTY720 increased ceramide levels in intact cells (see Supplementary Figure S5 at http://www.BiochemJ.org/bj/439/bj4390299add.htm). As FTY720 inhibits CerS (ceramide synthase) in vitro [47,48], we tested whether FTY720 increased ceramide production through a CerS-dependent mechanism. The CerS inhibitor Fumonisin B1 blocked the production of C16:0 ceramide, but not C22:0 or C24:0 ceramide, in response to FTY720 (Figure 6B). To complement these inhibitor studies, we performed C17-sphingosine labelling experiments. Endogenous sphingosine is an 18-carbon amino alcohol. By supplementing the culture medium with low levels of sphingosine with a 17-carbon backbone, C17-sphingosine metabolism to ceramide can be followed by MS due to the distinctive molecular mass of the products. This assay is an in situ CerS activity assay with the caveat that ceramidase running in reverse can also generate ceramide from the C17-sphingosine . Consistent with the selective decrease in C16:0 ceramide with Fumonisin B1 (Figure 6B), FTY720 treatment increased the amount of C17-sphingosine converted into C16:0 ceramide but not C22:0 or C24:0 ceramide (Figure 6C). Thus FTY720 increases ceramide levels via CerS-dependent and -independent mechanisms.
The effect of blocking ceramide production on FTY720-induced nutrient transporter loss and death was next determined. Fumonisin B1 provided limited protection from FTY720-induced transporter loss and cell death (Figures 6D and 6E), suggesting that C16:0 ceramide production contributes to, but is not essential for, these outcomes. To assess the contribution of C22:0 and C24:0 ceramide, we utilized Bax−/−Bak−/− cells. Although basal ceramide levels are within normal limits, cells lacking Bak fail to generate ceramide in response to acute stress . Bax−/−Bak−/− cells did not generate ceramide in response to FTY720 treatment (Figure 6F). Despite the lack of ceramide production, FTY720 triggered transporter loss and death (Figures 3G and 6G). Nutrient transporter down-regulation was somewhat blunted in Bax−/−Bak−/− cells (compare Figure 1A with Figure 6G). Thus, although not essential, ceramide generation may contribute to FTY720-induced nutrient transporter loss and cell death.
As ceramide generation could not completely explain the effects of FTY720 on transporters, we considered other mechanisms of action. CAT-1 is down-regulated in response to PKC (protein kinase C) activation by PMA [51,52]. We therefore tested whether FTY720 regulated transporter expression through the same mechanism. PMA reduced surface 4F2hc levels to a similar degree as FTY720 (Figure 7A). However, the PKC inhibitor bisindolylmaleimide I blocked PMA- but not FTY720-induced 4F2hc down-regulation. These results suggest that FTY720 decreases transporter levels through a parallel pathway. Consistent with this model, the effects of FTY720 and PMA on transporters were additive (Figure 7A). Ceramide and FTY720 activate PP2A (protein phosphatase 2A) [23,53]. PP2A activation has been proposed as part of the mechanism by which FTY720 kills cancer cells . Consistent with an important role for PP2A in FTY720-induced death, the PP2A inhibitor okadaic acid blocked nutrient transporter loss (Figures 7B and 7C). Bax−/−Bak−/− cells that failed to generate ceramide in response to FTY720 were also protected from transporter loss by okadaic acid (results not shown). These results implicate PP2A and to a lesser degree ceramide in FTY720-induced transporter loss.
FTY720-induced transporter loss is S1P receptor independent
FTY720 cannot be used in human cancer patients because the anti-neoplastic dose slows heart rate by activating S1P receptors [54,55]. However, the concentration of FTY720 required to trigger nutrient transporter loss is several logarithms above the EC50 for S1P receptors. We therefore predicted that nutrient transporter down-regulation was unrelated to and separate from S1P receptor activation. This idea was tested in several ways. FTY720 must be phosphorylated to activate S1P receptors . However, phosphorylated FTY720 did not affect 4F2hc surface expression, slow proliferation or kill cells (Figure 8A and results not shown). Similarly, pertussis toxin, which blocks a significant fraction of S1P receptor signalling , did not limit FTY720-induced transporter loss (Figure 8A). As a more definitive test, we evaluated AAL-149, an FTY720 analogue that does not activate S1P receptors because it is not phosphorylated [58–60]. AAL-149 down-regulated nutrient transporter proteins with identical kinetics and potency to FTY720 (Figure 8B) without affecting growth factor receptor-dependent signal transduction (Figures 4E and 4F). Consistent with a model where death is secondary to nutrient transporter loss, the cell-permeant nutrients oleic acid, dimethyl succinate and dimethyl α-oxoglutarate reduced AAL-149-induced death in non-transformed FL5.12 cells (Figure 8C). In keeping with the idea that oncogenic mutations may lock cancer cells into particular metabolic pathways, only dimethyl α-oxoglutarate supplementation protected Sup-B15 leukaemia cells (see Supplementary Figure S6A at http://www.BiochemJ.org/bj/439/bj4390299add.htm). Also consistent with toxicity secondary to intracellular nutrient limitation and bioenergetic stress, AAL-149 killed apoptosis-deficient Bax−/−Bak−/− cells as efficiently as FTY720 in a cell permeant nutrient-sensitive manner (Supplementary Figure S6B). Stress response hormesis was also observed for AAL-149; pretreatment with either 2-DG or rapamycin inhibited AAL-149-induced death (Figures 8D and 8E). These results with AAL-149 definitively demonstrate that the ability to induce nutrient transporter loss and cell death is separable from FTY720's dose-limiting toxicity.
Autophagy is a protective response to nutrient transporter loss (Figure 2F and Supplementary Figure S1B) and tumour cells are often autophagy-deficient . Thus AAL-149 could be particularly effective when combined with a drug that interferes with autophagy. By inhibiting lysosomal acidification, chloroquine blocks the degradation of autophagosomes, preventing nutrient liberation from the recycled material. Chloroquine is not a specific autophagy inhibitor, but it is a relatively safe drug that is already approved for use in humans and is being tested in multiple clinical trials in cancer patients . Combining cytostatic concentrations of AAL-149 and chloroquine killed all Sup-B15 leukaemia cells in the culture (Figures 9A and 9B). Synergy was also observed in PC3 prostate cancer cells (Figures 9C and 9D). Given these promising results in human cancer cell lines, AAL-149 and chloroquine were evaluated in colony assays with BCR-ABL-positive B-cell acute lymphoblastic leukaemia cells isolated from six different patients who had relapsed after therapy. AAL-149 blocked colony formation by these primary leukaemias as effectively as FTY720 (Figure 9E). As seen in cancer cell lines, combining AAL-149 with chloroquine produced synergy; in fact, adding chloroquine at just 1 μM was sufficient to reverse the resistance to 3 μM AAL-149 observed in one patient-derived sample that led to large error bars under this condition. As normal cells have a greater autophagic capacity, we hypothesized that AAL-149 might retain FTY720's selectivity for cancer cells even in combination with chloroquine. In fact, at concentrations that dramatically inhibited colony formation by leukaemia cells, AAL-149 had no effect on granulocyte-macrophage colony formation from normal peripheral blood mononuclear cells even when combined with chloroquine (Figures 9E and 9F). Together, these results strongly suggest that drugs that decrease nutrient transporter levels will be effective and selective anti-cancer agents, particularly in combination with autophagy inhibitors.
Drugs directed at almost every stage of biosynthesis are in or moving towards cancer clinical trials [62,63], but none target nutrient transporter proteins. The work we show in the present study suggests that sphingolipid-based drugs might be developed to trigger nutrient transporter loss and selectively starve cancer cells to death. Our previous studies indicated that ceramide down-regulates transporters for both amino acids and glucose and selectively kills highly anabolic cells . However, the challenges associated with using ceramide, an extremely hydrophobic molecule, therapeutically are still being overcome. In the present study we show that FTY720, a water-soluble orally bioavailable sphingolipid with favourable pharmacological properties limits nutrient transporter expression in vitro and in vivo. As FTY720 down-regulates transporters for both amino acids and glucose, the development of resistance by switching to alternative fuels might be limited. Moreover, diverse forms of cancer with distinct fuel preferences should be susceptible, particularly when transporter loss is combined with autophagy inhibition. Given their novel mechanism of action, compounds that trigger transporter loss might also be combined with current cancer therapies. These drug combinations might be effective even in aggressive or multidrug-resistant tumours such as the relapsed patient leukaemias evaluated in Figure 9(E).
One of the most striking findings is that drugs that profoundly down-regulate nutrient transporter proteins are highly selective for cancer cells and minimally toxic to normal primary cells (Figures 5D and 9F) . At the same time, non-transformed cell lines such as FL5.12 and MEFs are as sensitive to FTY720 and AAL-149 as their transformed counterparts. This apparent paradox can be explained by the fact that rapidly proliferating tissue culture-adapted cell lines have a metabolic profile very similar to cancer cells, exhibiting high rates of glycolysis and protein and membrane synthesis following adaptation to culture conditions where growth factors and nutrients are virtually unlimited [41,43,44]. Although these rapidly proliferating cell lines are not locked into their anabolic programme by oncogenic mutations, adaptive responses to acute transporter loss would have to be executed very quickly while the cells are under severe bioenergetic stress. When transporters are acutely down-regulated in these non-transformed cells proliferating at full speed, bioenergetic stress probably kills the cells before adaptive changes in metabolism can take place. Primary cells that are not growing as rapidly as FL5.12 cells or MEFs are likely to have more time to make adaptive changes to their metabolic programme before a bioenergetic crisis ensues. Consistent with this model, adapting FL5.12 cells to low growth factor or nutrient levels afforded significant protection from FTY720- or AAL-149-induced death (Figures 4A–4D, 8D and 8E).
FTY720 blocks cancer progression and metastasis in a wide variety of animal models, but its mechanism of action at the anti-neoplastic dose had not been determined. FTY720 was suggested by Neviani et al.  to kill cancer cells by activating the serine/threonine phosphatase PP2A which, through an undefined mechanism, was proposed to decrease the level of tyrosine phosphorylation of BCR-ABL and reduce BCR-ABL protein levels and signalling. However, many of the cancer cell lines that are sensitive to FTY720-induced cell death do not express BCR-ABL [19–22,24]. Moreover, we find that non-transformed, growth factor-dependent cells lines are just as sensitive to the drug as their BCR-ABL-transformed counterparts. The present study and previously published data [19–24] is instead consistent with a model where FTY720 kills cancer cells by inducing nutrient transporter loss and starvation. As in starving cells, reducing anabolism by pre-treating cells with rapamycin or 2-DG limits the cell death induced by FTY720 and AAL-149 (Figures 4C, 4D, 8D and 8E). These treatments do not protect cells from the loss of growth and survival signalling (Supplementary Figures S3A and S3B). On the other hand, disabling apoptosis efficiently blocks death in cells deprived of growth factor receptor signalling, but did not protect cells from FTY720 and AAL-149 (Figures 3E–3G and Supplementary Figure S6B). As cell-permeant nutrients rescued these apoptosis-deficient cells (Supplementary Figure S6B), fuel is the limiting factor. Our experiments using autophagy- or AMPK-deficient cells (Figures 2F and 2G, and Supplementary Figure S1B), cell-permeant nutrients (Figures 3A and 8C, and Supplementary Figure S6A) and an inhibitor of raft-dependent endocytosis (Figures 3B and 3C) also strongly support our proposal that bioenergetic stress subsequent to nutrient transporter loss is the principal mechanism behind the selective toxicity of FTY720 and AAL-149 for cancer cells. PP2A could trigger nutrient transporter loss through effects on the actin cytoskeleton, an important player in the clathrin-independent endocytic pathway utilized by these transporters . Alternatively, the Arf6 GTPase that controls the trafficking of these transporters is regulated by PP2A [31,65,66]. Now that we have uncovered this novel mechanism of action for FTY720 at the anti-neoplastic dose, future studies will further dissect the contributions of PP2A and Arf6 to sphingolipid- and AAL-149-induced nutrient transporter loss.
Although FTY720 is a selective and effective anti-neoplastic agent in animal models, dose-limiting bradycardia prevents its use in human cancer patients. AAL-149 does not activate S1P receptors, but down-regulates nutrient transporter proteins and selectively kills cancer cells with a similar potency to FTY720 (Figures 8 and 9). This result clearly demonstrates that the dose-limiting toxicity and anti-cancer effects of FTY720 are separate. Pharmacokinetic and toxicity studies with AAL-149 and other analogues with similar properties will be important next steps. FTY720 is rapidly metabolized to FTY720 phosphate in vivo; phosphorylated FTY720 does not reduce nutrient transporter expression or kill cells (Figure 8A). As AAL-149 is not phosphorylated, lower doses may produce a similar degree of transporter loss. On the other hand, structural modifications may be necessary to improve AAL-149's solubility or pharmacological properties. Other sphingolipids, including sphingosine itself, have similar effects on nutrient transporter proteins as FTY720-based drugs ( and results not shown). However, the therapeutic potential of these lipids is limited by their hydrophobicity and by their conversion into active metabolites such as S1P. S1P receptor-inactive FTY720 analogues are thus superior therapeutic candidates. In conclusion, our results from the present study indicate that nutrient transporter down-regulation could be a feasible, effective and novel approach to cancer therapy.
Kimberly Romero Rosales, Gurpreet Singh, Kevin Wu, Aimee Edinger and Eigen Peralta performed in vitro experiments with cell lines. Kimberly Romero Rosales and Matthew Janes executed the leukaemia studies in Figure 5. Michael Lilly performed the peripheral blood mononuclear cell experiments in Figure 9(F) and provided the human leukaemia samples to Matthew Janes who performed the colony assays in Figure 9(E). Leah Siskind performed experiments measuring ceramide levels in Bax−/−Bak−/− cells and helped to design the experiments evaluating the contribution of ceramide production to FTY720's ability to down-regulate transporters. Jie Chen, with input from Michael Bennett, quantified ceramide levels by MS. All authors were involved in planning experiments and data analysis. Aimee Edinger conceived the project, prepared the Figures, and wrote the paper. David Fruman edited the paper prior to submission and helped to design the in vivo experiments and those using primary human cells.
This work was supported by the National Institutes for Health [grant numbers F31 CA126494 (to K.R.R.), P20 RR17677, K08 CA100526 and R01 GM089919], the Gabrielle's Angel Foundation, the University of California, Cancer Research Coordinating Committee (UC CRCC), a Single Investigator Innovation Grant from the University of California, Irvine, Council on Research Computing and Libraries (UCI CORCL) to A.L.E., VA CDA-2, VA REAP and ACS [grant number IRG 97-219-11 (to L.J.S.)].
Abbreviations: AMPK, AMP-activated protein kinase; Bak, Bcl-2 homologous antagonist/killer; Bax, Bcl-2-associated X protein; Bcl-2, B-cell lymphoma 2; BCR, breakpoint cluster region; CAT-1, cationic amino acid transporter 1; CerS, ceramide synthase; DAPI, 4′,6-diamidino-2-phenylindole; 2-DG, 2-deoxy-D-glucose; FCS, fetal calf serum; 4F2hc, 4F2 heavy chain; GFP, green fluorescent protein; GLUT1, glucose transporter 1; IL-3, interleukin 3; MEF, murine embryonic fibroblast; mTOR, mammalian target of rapamycin; PKC, protein kinase C; PP2A, protein phosphatase 2A; S1P, sphingosine 1-phosphate; TCA, tricarboxylic acid
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