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Proc Natl Acad Sci U S A. Aug 9, 2011; 108(32): 13253–13257.
Published online Jul 25, 2011. doi:  10.1073/pnas.1110431108
PMCID: PMC3156152
Medical Sciences

Salinomycin inhibits Wnt signaling and selectively induces apoptosis in chronic lymphocytic leukemia cells

Abstract

Salinomycin, an antibiotic potassium ionophore, has been reported recently to act as a selective breast cancer stem cell inhibitor, but the biochemical basis for its anticancer effects is not clear. The Wnt/β-catenin signal transduction pathway plays a central role in stem cell development, and its aberrant activation can cause cancer. In this study, we identified salinomycin as a potent inhibitor of the Wnt signaling cascade. In Wnt-transfected HEK293 cells, salinomycin blocked the phosphorylation of the Wnt coreceptor lipoprotein receptor related protein 6 (LRP6) and induced its degradation. Nigericin, another potassium ionophore with activity against cancer stem cells, exerted similar effects. In otherwise unmanipulated chronic lymphocytic leukemia cells with constitutive Wnt activation nanomolar concentrations of salinomycin down-regulated the expression of Wnt target genes such as LEF1, cyclin D1, and fibronectin, depressed LRP6 levels, and limited cell survival. Normal human peripheral blood lymphocytes resisted salinomycin toxicity. These results indicate that ionic changes induced by salinomycin and related drugs inhibit proximal Wnt signaling by interfering with LPR6 phosphorylation, and thus impair the survival of cells that depend on Wnt signaling at the plasma membrane.

Keywords: canonical Wnt antagonist, LRP6 stability, B-cell neoplasm

Wnt signaling plays a crucial role in embryonic development and cancer (14). The Wnt/β-catenin signaling cascade is initiated when a Wnt molecule binds to the frizzled (Fzd) receptor and to the lipoprotein receptor-related protein 5 or 6 (LRP5/6) coreceptors, forming a ternary complex at the cell surface. The Fzds are seven-pass transmembrane receptors that have conserved extracellular cysteine-rich domains, which are responsible for Wnt–Fzd interactions. The LRP coreceptors are long single-pass transmembrane proteins that are also essential for Wnt-induced signal transduction (57). The formation of a Wnt/Fzd/LRP complex triggers phosphorylation of LRP5 or LRP6 at highly conserved PPPSPxP motifs in the intracellular domain. The phosphorylated LRP proteins recruit axin to the plasma membrane and undergo endocytosis. The eventual result is the inactivation of the β-catenin destruction complex, leading to β-catenin accumulation, nuclear translocation, and expression of Wnt target genes (8, 9).

Mutations and deletions in various components of the intracellular β-catenin destruction complex are common causes of human cancers (1, 10). However, other cancers without Wnt pathway mutations may depend upon exogenous Wnt signaling for survival. Included among the latter malignancies are chronic lymphocytic leukemia (CLL) and other B-cell neoplasms.

Increasing evidence also suggests that self-renewing stem-like cells within a tumor, which are resistant to conventional chemotherapy and are especially virulent, may also be controlled by Wnt/β-catenin signaling (11, 12). Thus, drugs targeted to the Wnt/β-catenin pathway may be useful as anticancer agents.

Salinomycin is a monocarboxylic polyether antibiotic widely used as an anticoccidiosis agent in chickens (13, 14). A recent study showed that salinomycin kills breast cancer stem cells at least 100 times more effectively than paclitaxel in mice (15). However, the mechanism by which salinomycin interferes with cancer stem cell survival is still unclear. In the present study, we identified salinomycin as an inhibitor of Wnt/β-catenin signaling, which acts on the Wnt/Fzd/LRP complex. The drug blocks Wnt-induced LRP6 phosphorylation, and causes degradation of the LRP6 protein. Treatment of primary CLL cells with salinomycin depressed LRP6 levels, down-regulated the expression of some Wnt target genes, and impaired cell survival. These results suggest that the anticancer effects of salinomycin may be at least partially attributable to Wnt inhibition.

Results

Salinomycin Suppresses Wnt1-Induced Signaling Pathway.

An initial screen of known drugs, performed by using the TOPflash cell reporter system (16, 17), identified the ionophores salinomycin, nigericin, thapsigargin, and ionomycin as inhibitors of the Wnt signaling pathway. To further characterize the Wnt inhibitory effect of salinomycin, HEK293 cells were transfected with the SuperTOPflash reporter and a Wnt1 expression plasmid. Expression of Wnt1 increased the transcriptional activity of the SuperTOPflash reporter more than 400 fold versus basal levels (Fig. 1A). Wnt1 overexpression also caused β-catenin accumulation in HEK293 cells (Fig. 1D). Salinomycin strongly suppressed Wnt1-stimulated reporter activity with an IC50 of 163 nM (Fig. 1A), and reduced β-catenin levels (Fig. 1D). In control experiments, salinomycin did not inhibit the luciferase activity of an AP1-Luc reporter, and actually enhanced the activity of an NFAT-Luc reporter (Fig. 1 B and C).

Fig. 1.
Salinomycin specifically inhibits the Wnt/β-catenin signaling pathway initiated by Wnt1. (A) HEK293 cells were transfected with a SuperTOPflash reporter and empty vector or Wnt1 expression plasmid. (B) An AP1-luciferase reporter was transfected ...

Effects of Salinomycin on Different Components of Wnt Signaling Cascade.

To determine the mechanism by which salinomycin inhibits Wnt/β-catenin signaling, the SuperTOPflash reporter was activated proximally by Wnt1or Fzd5/LRP6, or more distally by β-catenin, in transient transfection assays. Although nanomolar concentrations of salinomycin inhibited Wnt1-induced signaling, the drug had very little effect on reporter gene activity in cells that overexpressed Fzd5/LRP6 or β-catenin (Fig. 2 B and C). Much higher concentrations (4 μM) of salinomycin were needed to block the Wnt signaling induced by the downstream activators (Fig. 2D). Thus, salinomycin can suppress Wnt signaling by at least two different mechanisms, depending on drug dosage.

Fig. 2.
Salinomycin inhibits Wnt/β-catenin signaling via at least two different mechanisms. The SuperTOPflash reporter was transfected into HEK293 cells along with expression plasmids encoding Wnt1 (A), Fzd5 and LRP6 (B), and β-catenin (C), respectively. ...

Salinomycin Blocks Wnt1-Induced LRP6 Phosphorylation.

To test whether salinomycin directly targets the Wnt or LRP6 molecules, Wnt1, Wnt3 or dickkopf-related protein 2 (DKK2) were used to activate Wnt signaling. Although DKK2 can act as an agonist or antagonist of Wnt signaling, it has been shown to activate Wnt signaling when LRP6 is overexpressed (18). Wnt1, Wnt3, and DKK2 each enhanced the activity of the SuperTOPflash reporter, and these effects were antagonized by 250 nM salinomycin (Fig. 3). In contrast, salinomycin had much less effect on Fzd5/LRP6-mediated reporter gene activity in cells overexpressing Fzd/LRP6 (Fig. 3 A and B).

Fig. 3.
Salinomycin selectively targets the Wnt/Fzd/LRP complex. (A) Salinomycin selectively inhibited Wnt signaling mediated by Wnt1/Fzd5/LRP6 or Wnt3/Fzd5/LRP6 or DKK2/Fzd5/LRP6. The SuperTOPflash reporter assays were performed on HEK293 cells transfected with ...

Previous studies have demonstrated that Wnt pathway activation requires LRP6 phosphorylation by casein kinase-1γ, glycogen synthase kinase-3β, and various other kinases (8, 19). Indeed, nanomolar concentrations of salinomycin blocked Wnt1-induced LRP6 phosphorylation at serine 1490 in HEK293 cells, as assessed by immunoblotting with a specific antibody (Fig. 3C). Furthermore, salinomycin treatment caused degradation of the LRP6 protein in a dose-dependent fashion (Fig. 3C).

Nigericin, Like Salinomycin, Inhibits Wnt Signaling Cascade.

Nigericin is a potassium ionophore with structural similarity to salinomycin, which is also toxic to breast cancer stem cells (15). Nigericin, like salinomycin, selectively inhibited Wnt1-mediated signaling in HEK293 cells at nanomolar concentrations, but had little effect on reporter activity induced by β-catenin overexpression (Fig. 4). These results point to a common mechanism underlying the Wnt inhibitory action of the two potassium ionophores.

Fig. 4.
Comparison of the Wnt inhibitory effects of salinomycin, nigericin, and thapsigargin. HEK293 cells were cotransfected with the SuperTOPflash reporter construct, along with expression plasmids for Wnt1 (A), Fzd5/LRP6 (B), and β-catenin (C), respectively. ...

Effect of Calcium Ionophores.

Salinomycin can potentially disrupt Na+/Ca2+ exchange and cause an increase in cytoplasmic calcium levels (20, 21). It has been established that the canonical Wnt/LRP6-mediated signaling cascade is antagonized by elevating intracellular calcium levels (16). However, the calcium ionophores thapsigargin (Fig. 4) and ionomycin (Fig. S1) strongly inhibited Wnt pathway activation by Fzd5/LRP6 and, to a lesser degree, β-catenin, at concentrations at which salinomycin was ineffective.

Sensitivity of CLL Cells to Salinomycin.

Primary CLL cells produce Wnt proteins, have constitutive Wnt activation, and express high levels of the Wnt-regulated transcription factor Lymphocyte Enhancing Factor 1 (LEF1) (22). However, CLL cells do not have known activating mutations or deletions of the β-catenin transcription complex. Hence, CLL cell survival might be sensitive to compounds that inhibit proximal Wnt signaling by interference with LRP6 phosphorylation and stability. To test this hypothesis, primary CLL cells from 13 patients and peripheral blood mononuclear cells (PBMCs) from seven healthy donors were incubated with increasing concentrations of salinomycin for 48 h. Induction of apoptosis was detected by cytofluorometry. Incubation of the malignant lymphocytes with salinomycin induced apoptosis within 48 h, with a mean IC50 of 230 nM. Under the same conditions, salinomycin failed to induce apoptosis in PBMC at 100-fold higher concentrations (Fig. 5). This result suggests that salinomycin has selective cytotoxicity to CLL cells that are known to depend on Wnt signaling.

Fig. 5.
Salinomycin selectively induced apoptosis in CLL cells. Primary CLL cells from 13 patients and the PBMCs from seven normal donors were treated with increasing amounts of salinomycin for 48 h. Apoptosis was measured by flow cytometry by using DiOC6/PI ...

Salinomycin Reduces LRP6 Protein Levels and Down-Regulates Expression of Wnt Target Genes in CLL Cells.

LRP6 expression has been detected in primary CLL cells, albeit at low levels (22, 23). As salinomycin could block Wnt-induced LRP6 phosphorylation and cause the degradation of LRP6 protein in HEK293 cells (Fig. 3C), we also assayed its effect on the LRP6 protein in CLL cells. As predicted, the drug reduced LRP6 levels in the malignant lymphocytes (Fig. 6A).

Fig. 6.
Salinomycin reduced the LRP6 protein level and down-regulated the expression of LEF1, cyclin D1, and fibronectin in CLL cells. (A) Primary CLL cells from two patients were treated with indicated amounts of salinomycin for 16 h. The phospho-LRP6 (Ser1490), ...

LEF1, cyclin D1, and fibronectin are target genes of the Wnt/β-catenin pathway, which are up-regulated in CLL cells compared with normal lymphocytes (22, 24). The CLL cells from four patients were treated with salinomycin for 16 h, and then were analyzed by quantitative PCR for gene expression. At this time point, the CLL cells were still viable, but all three Wnt-dependent transcripts had decreased (Fig. 6B).

Discussions

Salinomycin and nigericin are among the first drugs reported to act as selective cancer stem cell inhibitors (15). These polyether ionophores interfere with transmembrane potassium potential and promote mitochondrial and cellular potassium efflux (25, 26). Our experiments demonstrate that salinomycin treatment of cells antagonizes the Wnt signaling cascade. At submicromolar concentrations, the drug inhibits Wnt1-induced LRP6 phosphorylation, and causes the degradation of the LRP6 protein, an essential component of the Wnt receptor complex. At micromolar concentrations, salinomycin also promotes β-catenin degradation. Considering the importance of Wnt/β-catenin signaling in stem cell biology and in various malignancies, the Wnt antagonistic actions of salinomycin may contribute to its toxicity toward breast cancer progenitor cells.

The phosphorylation of LRP6 is crucial for activation of Wnt/β-catenin signaling. Several protein kinases, including glycogen synthase kinase-3 and casein kinase-1γ, have been shown to phosphorylate this Wnt coreceptor (27). A recent study showed that transmembrane protein 198 can promote LRP6 phosphorylation and Wnt signaling activation by recruitment of casein kinase family proteins (28). Another report showed that salinomycin is able to overcome multidrug resistance mediated by the ATP-binding cassette transporter p-glycoprotein 170 (29). Cruciat et al. demonstrated that LRP6 phosphorylation and internalization requires the activity of the vacuolar H+-adenosine triphosphatase (V-ATPase) (30). The prorenin receptor functions as a specific adaptor between LRP6 and V-ATPase. The V-ATPase protein complex localizes in intracellular organelles and at the plasma membrane, where it pumps protons and acidifies vesicles, thereby promoting endocytosis (31). Inhibition of V-ATPase activity by bafilomycin blocks Wnt/β-catenin signaling (30). Nigericin has been reported to induce intracellular alkalinization (32, 33). It is therefore likely that salinomycin and nigericin may interfere with the proton gradient generated by the V-ATPase, and thus block the phosphorylation and endocytosis of LRP6 that are required for initial Wnt signal transduction.

Previous studies have revealed that salinomycin can also cause an increase in intracellular calcium levels by disrupting Na+/Ca2+ exchange (21). The toxicity of salinomycin has been attributed to drug-induced accumulation of intracellular calcium (20). As an increase in intracellular calcium causes inhibition of the canonical Wnt pathway (16), we tested the effect of other intracellular calcium regulators on LRP6-mediated Wnt signaling. Unlike salinomycin, both thapsigargin and ionomycin blocked Wnt signaling downstream of LRP6. Thus, at least at submicromolar concentrations, salinomycin-induced inhibition of Wnt signaling probably does not depend on its calcium mobilizing activity.

The LRP5 and LRP6 coreceptors for Wnt signaling have been revealed as potential oncogenic proteins. Aberrant expression of an LRP5 splicing isoform has been implicated in breast tumor formation (34), and LRP6 is overexpressed in a subset of human breast carcinomas (35). Overexpression of LRP6 in the mouse mammary gland induces mammary hyperplasia, whereas down-regulation of LRP6 inhibits breast cancer tumorigenesis (35, 36). Furthermore, an LRP antagonist, Mesd (mesoderm development), markedly suppresses breast cancer tumor growth in vivo without significant toxicity (35, 36). These studies strongly suggest that LRP6 is a potential therapeutic target in breast cancer. The fact that salinomycin can block Wnt-induced LRP6 phosphorylation and cause degradation of LRP6 protein, indicates that LRP6 is a “druggable” target for anticancer therapy. Small-molecule drugs that alter LRP6 phosphorylation or stability may have therapeutic potential in malignancies with abnormal Wnt activation at the cell surface. They would be expected to be less active in cancers with mutational activation of the β-catenin destruction complex, such as colorectal carcinomas.

CLL is the most frequent form of human leukemia in the Western world. Several activating Wnt proteins are overexpressed in primary CLL cells, whereas physiological inhibitors of Wnt signaling are partly inactivated in this disease (22, 37). During Wnt activation, β-catenin is up-regulated and cooperates with the transcription factors T-cell factor and LEF1 to alter the production of proteins important for cell proliferation and survival, including Myc, cyclin D1, and several antiapoptotic proteins. In comparison with normal blood B cells, LEF1 is the most highly up-regulated mRNA in CLL cells (38). Knockdown of LEF1 by siRNA leads to increased apoptosis of CLL cells (39, 40). Treatment with two small-molecule inhibitors of Wnt/β-catenin signaling (CGP049090 and PKF115-584) induces apoptosis in CLL cells in vitro and in vivo (39). Moreover, our previous results revealed that ethacrynic acid and other Michael acceptors exhibit selective toxicity to CLL cells, in part by antagonism of Wnt/β-catenin signaling (17, 41). In the present study, we demonstrated that salinomycin reduces phospho-LRP6 and total LRP6 protein levels, whereas a higher concentration (more than 500 nM) of salinomycin is required for its maximum effect on LRP6 phosphorylation and stability in unmanipulated CLL cells at earlier time points. Interestingly, we observed that 250 nM salinomycin was a high enough dose to block LRP6 phosphorylation induced by overexpressing Wnt1 in HEK293 cells. Considering multiple kinsaes responsible for LRP6 phosphorylation (27), our results suggest that salinomycin may specifically suppress Wnt-induced LRP6 phosphorylation. Furthermore, we showed that salinomycin down-regulates the expression of Wnt target genes and exerts selective toxicity to primary CLL cells compared with normal PBMCs. Thus, drugs that inhibit LRP6 phosphorylation or stability may be therapeutically active in CLL.

Materials and Methods

Human Samples.

Samples were collected by the CLL Research Consortium after informed consent was obtained from patients fulfilling diagnostic criteria for CLL. Institutional review board approval was obtained from the University of California, San Diego, for the procurement of patient samples in this study, in accordance with the Declaration of Helsinki. Buffy coat samples from healthy volunteers were obtained from the San Diego Blood Bank.

Reagents and Plasmids.

Salinomycin, nigericin, thapsigargin, and ionomycin were purchased from Sigma-Aldrich. A Gen-plus collection of 960 known drugs was obtained from Microsource. The reporter plasmid TOPflash and the β-catenin expression plasmid were gifts from H. Clevers (University of Utrecht, Utrecht, The Netherlands). The SuperTOPflash reporter construct was from Karl Willert (University of California, San Diego, San Diego, CA). The NFAT-Luc and AP1-Luc reporters were purchased from BD Biosciences. The expression plasmid for human DKK2 was from Origene Technologies. The expression plasmids encoding Wnt1, Wnt3, LRP6, NFATc, H-rasV12, and β-gal have been described previously (22, 42, 43).

Cell Culture and Transfection.

HEK293 cells were maintained in DMEM supplemented with 10% FBS and 100 μg/mL penicillin and 100 μg/mL streptomycin. HEK293 cells were transfected in 12-well plates by using FuGENE (Roche), and 0.5 μg of reporter plasmid, 50 to 100 ng of the control plasmid pCMXβgal, 100 to 200 ng of the various expression plasmids, and carrier DNA pcDNA3 plasmid, for a total of 1 μg per well. After overnight incubation, the cells were treated with the different concentrations of drugs or vehicle. For luciferase assays, cells were lysed in isotonic potassium phosphate buffer, pH 7.8, containing 1% Triton X-100, and luciferase activities were assayed in the presence of substrate by using a multiple-mode detection reader (Infinite M200; Tecan). The luciferase values were normalized to the activity of the β-gal, and are expressed as percentage of control luciferase or fold stimulation of luciferase activity compared with the basal level. All of the transfection results are representative of a minimum of three independent transfections.

Western Blot Analysis.

HEK293 cells and primary CLL cells were treated with the indicated amounts of salinomycin as described in the figure legends. Cells were washed twice with PBS solution and resuspended in 0.5 mL lysis buffer (20 mM Tris-HCl, pH 8.0/10% glycerol/5 mM MgCl2/0.15 M KCl/0.1% Nonidet P-40 with protease inhibitors). Equal amount of proteins from each sample was resolved by SDS/PAGE followed by immunoblotting with anti-LRP6 antibody, phospho-LRP6 (Ser1490) antibody (Cell Signaling Technology), anti–β-catenin antibody (Santa Cruz Biotechnology), and β-actin (Chemicon International). Horseradish peroxidase-conjugated anti-IgG was used as the secondary antibody. The membranes were developed by using a chemiluminescence system (ECL detection reagent; Amersham Pharmacia).

RNA Isolation and Real-Time PCR.

Total RNA was isolated from 1 × 106 primary CLL cells by TRIzol reagent (Invitrogen). The RNA samples were further purified by using a RNeasy Protect kit (Qiagen). The mRNA levels were quantified in triplicate by real-time PCR on a 7900HT Fast Real-Time PCR System (Applied Biosystems) by using the following primer sets to detect the expression of LEF1, cyclin D1, and fibronectin. LEF1 sense, 5′-AGGAACATCCCCACACTGAC-3′; and LEF1 antisense, 5′-AGGTCTTTTTGGCTCCTGCT-3′. Cyclin D1 sense, 5′-AATGACCCCGCACGATTTC-3′; and cyclin D1 antisense, 5′-TCAGGTTCAGGCCTTGCAC-3′. Fibronectin sense, 5′-ACCTACGGATGACTCGTGCTTT-3′; and fibronectin antisense, 5′-TTCAGACATTCGTTCCCACTCA-3′. PCR was performed by using Power SYBR Green PCR Master Mix (Applied Biosystems) according to the manufacturer's instructions. The PCR conditions were 2 min at 50 °C (one cycle), 10 min at 95 °C (one cycle), and 15 s at 95 °C and 1 min at 60 °C (40 cycles). The data were analyzed by using the comparative Ct method, where Ct is the cycle number at which fluorescence first exceeds the threshold. The ΔCt values from each tissue were obtained by subtracting the values for 18S Ct from the sample Ct. One difference of Ct value represents a twofold difference in the level of mRNA.

Cell Apoptosis Assays.

Apoptosis was determined by the analysis of mitochondrial transmembrane potential (ΔΨm) using 3,3′-dihexyloxacarbocyanine iodine (DiOC6) and by cell membrane permeability to propidium iodide (PI). Primary CLL cells were cultured at a density of 5 × 106 cells/mL in RPMI with 10% FBS containing salinomycin at concentrations ranging from 0 to 10 μM. After treatment for 48 h, 100 μL of the cell culture was collected and transferred to FACS tubes containing 100 μL FACS buffer with 60 nM DiOC6 and 10 μg/mL PI. Cells were incubated at 37 °C for 30 min and analyzed within 30 min by flow cytometry by using a FACSCalibur (Becton Dickinson). Fluorescence was recorded at 525 nm (FL-1) for DiOC6 and at 600 nm (FL-3) for PI. The apoptotic cells were determined by calculating the percentages of the DiOC6/PI+ CLL populations.

Supplementary Material

Supporting Information:

Acknowledgments

We thank Laura Rassenti for technical assistance and Nancy Noon for secretarial support. T.J.K. and D.A.C. are members of the Chronic Lymphocytic Leukemia Research Consortium. This work was supported in part by Leukemia and Lymphoma Society SCOR Grant CA81534-06, National Cancer Institute–Drug Development Group Grant CA113318-01, and National Institutes of Health Grant PO1-CA081534 (to D.A.C.).

Footnotes

The authors declare no conflict of interest.

This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.1073/pnas.1110431108/-/DCSupplemental.

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