Targeting Wnt/β-catenin by anthelmintic drug niclosamide overcomes paclitaxel resistance in esophageal cancer

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ABSTRACT

Esophageal cancer is an aggressive malignancy and its current treatment strategies are plagued with high rates of recurrence. In this work, we demonstrate that niclosamide, an anthelmintic drug, is a potential sensitizing candidate for overcoming chemoresistance in esophageal cancer. Using a panel of esophageal cancer cell lines and normal cells, we show that niclosamide has anti-esophageal cancer activity and is likely to be less effective against normal esophageal epithelial and fibroblast cells. The combination of niclosamide with paclitaxel results in much greater efficacy than paclitaxel alone, suggesting that niclosamide is active against esophageal cancer cells that are resistant to paclitaxel. This is further confirmed by our results that niclosamide is effective in inhibiting proliferation and inducing apoptosis in paclitaxel-resistant esophageal cancer cells. In line with the findings obtained from in vitro cell culture system, niclosamide augments the in vivo efficacy of paclitaxel and significantly arrests paclitaxel-resistant esophageal cancer growth without causing toxicity in mice. Mechanistically, we show that niclosamide decreases β-catenin level and activity, and inhibits phosphorylation of STAT3 and mTORC1 substrate 70S6K. Stabilization of β-catenin level by Wnt activator lithium chloride (LiCl) significantly abolishes the inhibitory effects of niclosamide in inhibiting proliferation and survival but not suppressing phosphorylation of STAT3 and 70S6K in paclitaxel-resistant esophageal cancer cells, suggesting that niclosamide sensitizes esophageal cancer cell to paclitaxel mainly through inhibiting Wnt/β-catenin. Our work demonstrates the efficacy of niclosamide and its underlying mechanism in paclitaxel-resistant esophageal cancer. Our work emphasizes that Wnt/β-catenin inhibition is a sensitizing strategy in esophageal cancer.

Key words: niclosamide, esophageal cancer, paclitaxel-resistance, Wnt/β-catenin

INTRODUCTION

Esophageal cancer is one of the leading causes of cancer-related mortality with increasing incidence in recent years [1]. The major types of esophageal cancer are squamous cell carcinoma (~ 70%) and adenocarcinoma (~ 30%). Despite substantial advances in diagnostics and therapeutics that include surgical and chemoradiation advancement, the five-year survival rate is as low as ~20% for esophageal cancers. That has remained unchanged for several decades [2]. Whole genome sequencing of related samples identify mutations of TP53, CDKN2A, FAT1, NOTCH1, PIK3CA KMT2D, CUL3, ZFP36L2 and NEF2LF that may contribute to esophageal carcinogenesis and progression [3]. Besides driver DNA mutations, Wnt/β-catenin is activated in esophageal cancer and plays an important role in esophageal cancer development and radioresistance [4-6].

Niclosamide, a well-tolerated anthelmintic drug, is especially effective against cestodes [7]. Niclosamide kills cestodes via inhibiting oxidative phosphorylation and stimulating adenosine triphosphatase activity in the mitochondria of cestodes [8]. Interestingly, several high-throughput screenings consistently identified niclosamide as a potential anti-cancer agent [9-12]. Niclosamide potently inhibits multiple biological activities related to a number of human cancers, such as colorectal, ovarian cancers, leukemia and myeloma, and the list of niclosamide-targeted cancers is increasing [11, 13-16]. Apart from being effective as single drug alone, niclosamide significantly augments the efficacy of chemotherapeutic drugs (eg, cisplatin) and other anti-cancer drugs (eg, dasatinib) in pre-clinical cancer models [16-18]. Notably, the anti-cancer activity of niclosamide is attributed to its inhibition of multiple intracellular signalling pathways that are either over- expressed, constitutively active or mutated in many cancers, including Wnt/β-catenin, mTORC1, Stat3, NK-ĸB and Notch [19].

In this work, we systematically investigated in vitro and in vivo efficacy of niclosamide as single drug as well as its combination with paclitaxel (the first line chemotherapeutic drug for metastatic esophageal cancer [20]) on a panel of human esophageal cancer cell lines. We further challenged niclosamide on paclitaxel-resistant esophageal cancer cells and determined whether niclosamide’s effects are selective towards cancer cells by including human normal esophageal epithelial cells and fibroblast cells. Mechanism studies of niclosamide’s action in paclitaxel-resistant esophageal cancer cells were performed focusing on Wnt/β-catenin, mTORC1 and Stat3.

MATERIALS AND METHODS

Cells culture, generation of chemoresistant cell lines and drugs

Human esophageal cancer cell lines ESO26, FLO-1, KYAE-1, OE33, SK-GT-4 and OE19 (ECACC, UK) were cultured in Dulbecco’s Modified Eagle Medium (DMEM) supplemented with 2mM glutamine, 10% fetal bovine serum (FBS) and 1% penicillin/streptomycin. The authenticity of the six human esophageal cancer cell lines selected in our study has been verified [21]. Immortalized human normal esophageal epithelial cell line NE2-hTERT (Abm, US) was cultured in 1:1 ratio of Defined Keratinocyte-SFM (Gibco, #10744-019). Immortalized human normal fibroblast cell line BJ-5ta was cultured in DMEM supplemented with 4 mM L-glutamine, 4.5 g/L glucose and 1.5 g/L sodium bicarbonate. ESO26-PAC-r and KYAE-1-PAC-r cells were established by prolonged exposure of parental cells to paclitaxel. The dose of paclitaxel started with 0.5 nM and was increased by 1.5-fold every two-three weeks. The next dose was given until the cells were stable in proliferation without significant death. ESO26-PAC-r and KYAE-1-PAC-r were maintained in the presence of 100 nM and 200 nM of paclitaxel, respectively. Niclosamide, lithium chloride (LiCl) and paclitaxel (Sigma, US) were reconstituted in DMSO and stored in – 200C.

Cell proliferation assay

1×104 cells/well in a 96-well plate were treated with niclosamide, paclitaxel or the combination of both for 3 days. BrdU (Bromodeoxyuridine / 5-bromo-2′-deoxyuridine) was added for 3 hours in the culture medium, and cell proliferation activity was determined using the BrdU Cell Proliferation ELISA kit (Abcam, US).

Cell viability assay

1×104 cells/well in a 96-well plate were treated with varying concentrations of paclitaxel for 3 days. Cell viability were determined using the MTT (3-(4,5-dimethylthiazol-2-yl)-2,5- diphenyltetrazolium bromide) Assay Kit (Abcam, US).

Cell apoptosis assay

1×106 cells/well in a 6-well plate were treated with niclosamide, paclitaxel or the combination of both for 3 days. Apoptotic cells were stained with FITC Annexin V Apoptosis Detection Kit with 7-AAD (BioLegend, USA) and determined by flow cytometry on MACSQuant® Analyzer.

Denaturing sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE) and Western blot (WB) analyses

1×107 cells in a 5cm petri dish were treated with niclosamide for 24 hours. Cells were lysed and proteins were extracted using 4% SDS. Protein content was determined using the bicinchoninic acid protein assay kit (Thermo Scientific, US). Proteins were resolved using denaturing SDS– PAGE and then analyzed by WB using designated primary and secondary antibodies (Santa Cruz Biotechnology, US). Immunoblots shown in the accompanying figures are the representative of three independent experiments.

Luciferase reporter assay

1×104 cells/well in 96-well plate were co-transfected with 30 ng/well M50 Super 8x TOPFlash plasmid DNA (a kind gift from Dr. Randall Moon) [22] with 3 ng/well pRL-TK Renilla luciferase plasmids DNA (Promega, US). After 24 hours post-transfection, cells were treated with niclosamide for 24 hours prior to performing the luciferase reporter assay (Promega, US) according to manufacturer’s instructions.

Esophageal cancer xenograft in SCID mice

The animal experiments were approved by the Institutional Animal Care and Use Committee of Hubei University of Arts and Science. SCID mice (Biocytogen Inc, China) were subcutaneously injected with 5 million oesophageal cancer cells. After the development of palpable tumors, the mice were randomized into different groups receiving drug treatment (drug dose and administration routes were specifically indicated in Figure Legends). After 3 weeks of drug treatment, mice were euthanized. Tumor length and width were measured using a calliper and calculated using the formula: length x (width)2/2.

Statistical analyses

Data are expressed as mean and standard deviation (SD). ANOVA was performed in figures 1 and 2, where comparisons of multiple categorical variables were made. Student t test was performed in figures 3 and 4 to make direct references to different conditions. ANOVA with Tukey’s Honest Significant Difference (HSD) post hoc testing was conducted. P values < 0.05 is considered statistically significant.

RESULTS

Niclosamide inhibits proliferation and survival of esophageal cancer cells

To determine whether niclosamide has selective anti-esophageal cancer activity, we conducted cell growth and survival assays using niclosamide on both esophageal cancer and normal cells including immortalized normal esophageal epithelial as well as fibroblast cells. Six human esophageal cancer cell lines in our studies cover a variety of cell types (eg, epithelial and squamous), cellular origins (eg, established from diverse patient tissues) and DNA driver mutations. As shown in Fig. 1A, niclosamide significantly inhibited proliferation on all tested esophageal cancer cell lines as assessed by BrdU incorporation method. Niclosamide also significantly induced apoptosis as shown by the increased Annexin V percentage in esophageal cancer cells (Fig. 1B). In contrast, niclosamide at the same concentrations did not inhibit immortalized normal esophageal epithelial cell or fibroblast in a less extent than esophageal cancer cells (Fig. 1). These results demonstrate that niclosamide is likely to be more effective in inhibiting esophageal cancer cells than normal cells.

Niclosamide is active against paclitaxel-resistant esophageal cancer cells in vitro

We asked whether niclosamide is effective in targeting chemo-resistant esophageal cancer cells. Paclitaxel is the first-line treatment for metastatic esophageal cancer [20]. We first performed combination studies to determine whether niclosamide enhances the anti-proliferative and pro- apoptotic effects of paclitaxel in esophageal cancer cells. If niclosamide is effective in targeting esophageal cancer cells that are resistant to paclitaxel, the combination of niclosamide and paclitaxel will achieve greater efficacy than paclitaxel alone. To observe the possible synergistic effects of the combination, the concentration of niclosamide and paclitaxel used in the combination studies was sublethal that inhibited proliferation and induced apoptosis by ~ 30% as a single drug alone (Fig. 2A and B). However, the combination led to almost complete inhibition of growth and survival in all tested esophageal cancer cell lines (Fig. 2A and B).

We then directly examined the effects of niclosamide on paclitaxel-resistant esophageal cancer cells. We established the paclitaxel-resistant ESO26-PAC-r and KYAE-1-PAC-r cells by prolonged exposure of gradually increasing concentrations of paclitaxel. We showed that ESO26- PAC-r and KYAE-1-PAC-r exhibited significantly higher resistance to paclitaxel, with IC50 of paclitaxel in resistant cells at least 10-fold higher than that in parent cells (Fig. 2C and D). In line with the combination studies, niclosamide significantly inhibited proliferation and induced apoptosis in ESO26-PAC-r and KYAE-1-PAC-r cells in a dose-dependent manner (Fig. 2E and F). Taken together, these results demonstrate that niclosamide is active against paclitaxel-resistant esophageal cancer cells.

Niclosamide overcomes paclitaxel-resistance in esophageal cancer cells mainly through inhibiting Wnt/β-catenin signaling

A number of essential signaling pathways have been identified as the target of niclosamide in cancer cells, including Wnt/β-catenin, STAT3 and mTORC1 [19]. Given their important roles in cancer cell chemoresistance, we performed immunoblot analysis to determine whether niclosamide affects the expression or phosphorylation levels of the molecules involved in Wnt/β- catenin, STAT3 and mTORC1 signaling in paclitaxel-resistant esophageal cancer cells. We found that niclosamide downregulated Dvl2 and β-catenin levels in ESO26-PAC-r and KYAE-1-PAC-r cells (Fig. 3A). In addition, phosphorylation of STAT3 at Tyr705 and phosphorylation of mTORC1 substrate 70S6K at Thr389 were inhibited by niclosamide (Fig. 3A), suggesting that niclosamide inhibits STAT3 and mTORC1 signaling pathways.

TOPflash assay analysis further demonstrated that niclosamide inhibited the activation of Wnt/β- catenin pathway in a dose-dependent manner in paclitaxel-resistant esophageal cancer cells (Fig. 3B). Wnt activator LiCl which activates Wnt signalling via inhibiting β-catenin degradation [23]
reversed the effect of niclosamide in decreasing β-catenin but not phosphorylation of STAT3 and S6K (Fig. 3C). Notably, niclosamide was significantly less effective in inhibiting proliferation and inducing apoptosis of ESO26-PAC-r and KYAE-1-PAC-r cells in the presence of LiCl (Fig. 3D and E). These suggest that niclosamide overcomes paclitaxel-resistance in esophageal cancer cells mainly through inhibiting Wnt/β-catenin signalling.Wang et al’s work has reported that niclosamide-induced Wnt signaling inhibition in colorectal cancer is mediated by autophagy, most likely by inhibited mTORC1 signaling [24]. To understand whether autophagy or mTORC1 signaling is involved in the inhibition of Wnt signalling by niclosamide in esophageal carcinoma cells, we treated cells with the presence of niclosamide with 3-Methyladenin (3MA, autophagy inhibitor) or rapamycin (mTORC1 inhibitor). The concentrations of 3MA and rapamycin used in the study are referred from the previous work demonstrating the effective dose of inhibiting autophagy or mTORC1 signalling in cells [24, 25]. We also confirmed their inhibitory effects on autophagy or mTORC1 signalling in ESO26-PAC-r cells (Supplementary Fig. 1A). We further found that neither 3MA nor rapamycin affected the inhibitory effect of niclosamide on Wnt/β-catenin activity in ESO26-PAC-r cells (Supplementary Fig. 1B). This demonstrates that niclosamide inhibits Wnt/β-catenin in a mTORC1-independent manner.

Niclosamide is active against paclitaxel-resistant esophageal cancer cells in vivo

To determine the translational potential of niclosamide in esophageal cancer, we investigated the in vivo efficacy of niclosamide using two independent esophageal cancer xenograft mouse models. Paclitaxel-sensitive and -resistant models were generated by subcutaneously implanted ESO26 and ESO26-PAC-r cells into immunodeficient mice, respectively. Drug treatment was initialized after the development of palpable tumors and tumor size was monitored throughout the duration of treatment. Mice tolerated well with niclosamide, paclitaxel or the combination of both at the given doses as we did not observe any significant weight loss or abnormal appearance or behaviour of the mice (data not shown).

Consistent with the findings obtained from in vitro cell culture system, niclosamide or paclitaxel as single drug modestly inhibited ESO26 tumor growth, but the tumors continued to increase in size (Fig. 4A). The combination completely arrested tumor growth throughout the duration of treatment. In addition, niclosamide significantly inhibited ESO26-PAC-r tumor growth in mice (Fig. 4B). These results correlate well with each other, demonstrating that niclosamide is active against paclitaxel-resistant esophageal cancer cells in vivo.

DISCUSSION

Patients with advanced esophageal cancer develop resistance to chemotherapy and may relapse within a year [26]. More effective and selective therapies are required for better clinical management of esophageal cancer. Combination therapy consisting of chemotherapeutic drug and other agents with different molecular targets is a promising approach to minimise toxicity as well as maximize potency [27]. Identification of new indications for existing drugs has advantages than conventional drug development due to the known pharmacokinetics and safety profiles of existing drugs, and has been hotly investigated in major drug discovery programs [28]. In line with these efforts, our work is the first to demonstrate that niclosamide is an attractive candidate to overcome chemoresistance in esophageal cancer.

In agreement with the previous findings on the anti-cancer activities of niclosamide [29-33], we demonstrate that niclosamide is active against esophageal cancer cells regardless of cellular origin, cell type and genetic mutations (Fig. 1). In addition, we show that niclosamide at low micromolar concentrations exhibits preferential inhibitory effects on esophageal cancer cells compared to normal immortalized esophageal epithelial and fibroblast cell lines (Fig. 1), suggesting the safe therapeutic window of niclosamide in the treatment of esophageal cancer. The normal cells used in our study are immortalized cell lines. Primary normal cells are ideal model to serve as normal control to determine the selectivity of niclosamide. Nevertheless, the safety and efficacy of niclosamide is further validated in two independent esophageal cancer xenograft mouse models (Fig. 4). Our findings correlate well with previous work that niclosamide induces apoptosis of leukemia and prostate cancer cells but not normal cells [12, 16, 34], and niclosamide treatment in mice does not cause toxicity in vital organs (eg, liver, kidney and bone marrow) and normal tissues [35]. Our pre-clinical findings together with the previous studies clearly indicate that niclosamide at effective doses target cancer cells with minimal toxicity to non-cancerous cells.

A significant finding of our work is the identification of the ability of niclosamide to overcome chemoresistance in esophageal cancer as 1) niclosamide augments paclitaxel’s efficacy (Fig. 2A and B, and Fig. 4A) and 2) niclosamide is active against paclitaxel-resistant esophageal cancer cells in vitro and in vivo (Fig. 2C to D and Fig. 4B). A number of studies have stated the ability of niclosamide to overcome cancer chemoresistance by showing the enhanced combinatory effects of niclosamide with anti-cancer agents [17, 31, 35]. To our best knowledge, we are the first to demonstrate the direct inhibitory effects of niclosamide on the chemo-resistant cancer cells.

Niclosamide decreases the level of Dvl2 and β-catenin, and phosphorylation of STAT3 and mTORC1 substrate 70S6K in paclitaxel-resistant esophageal cancer cells (Fig. 3A), suggesting that niclosamide inhibits Wnt/β-catenin, STAT3 and mTORC1 signalings. These have been demonstrated as the targets of niclosamide in cancer cells [19]. However, we show that niclosamide inhibits paclitaxel-resistant esophageal cancer cells mainly through Wnt/β-catenin pathway because the stabilization of β-catenin remarkably reverses the inhibitory effects of niclosamide (Fig. 3C to E). mTORC1 has been suggested to be involved in the inhibitory effects of niclosamide in Wnt/ β-catenin in colorectal cancer cells [24]. Our study demonstrates that the inhibitory effect of niclosamide in Wnt/ β-catenin in esophageal cancer cells is mTORC1 signaling-independent (Supplementary Fig. 1). We and others suggest that the underlying mechanisms of niclosamide’s action in cancer cells are cell type-specific. Wnt/β-catenin is important in esophageal cancer progression and radioresistance [4, 6, 36]. Our work suggests that Wnt/β-catenin also plays roles in chemoresistance in esophageal cancer. Besides tumor bulk cells, niclosamide has been reported to target cancer stem cells [9, 34]. Given the role of Wnt/β-catenin in cancer stem cell biology [37], it would be interesting to investigate whether niclosamide is active against esophageal cancer stem cells.

In summary, our results show that niclosamide targets not only parental but also paclitaxel- resistant esophageal cancer cells in vitro and in vivo with minimal toxicity in normal cells, mainly via inhibiting Wnt/β-catenin. Our work provides pre-clinical evidence to initialize the clinical trials to evaluate the efficacy and safety of niclosamide in esophageal cancer patients, particularly those who develop chemoresistance.

DISCLOSURE OF CONFLICT OF INTEREST

None.

ACKNOWLEDGEMENT

This work was supported by a research grant provided by Hubei University of Arts and Science (Grant No. 661615).

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FIGURE LEGENDS
Fig. 1. Niclosamide inhibits proliferation and induces apoptosis of a panel of cultured human esophageal cancer cells. Niclosamide at 1, 2.5, 5 and 10 μM dose-dependently decreases proliferation (A) and increases apoptosis (B) in six esophageal cancer cell lines: SK-GT-4, FLO-1, OE19, KYAE-1, OE33 and ESO26. Niclosamide inhibits growth and induces apoptosis in normal esophageal epithelial cell NE2-hTERT and fibroblast BJ-5a in a less extent than esophageal cancer cells. Niclosamide at 0 nM was used in the control group for each cell line. Results shown are the fold change relative to control and obtained from at least three-independent experiments with triplicate. *p<0.05, compared to control.

Fig. 2. Niclosamide is effective in targeting paclitaxel-resistant esophageal cancer cells in vitro. Combination of niclosamide and paclitaxel (5 nM) is significantly more efficient in inhibiting proliferation (A) and inducing apoptosis (B) in esophageal cancer cells. Niclosamide at
2.5 μM was used in SK-GT-4, FLO-1 and OE19 cells in combination studies. Niclosamide at 1 μM was used in KYAE-1, OE33 and ESO26 for combination studies. ESO26-PAC-r (C) and KYAE-1-PAC-r (D) cells are more resistant to paclitaxel than ESO26 and KYAE-1 cells. Niclosamide at 5, 10 and 20 μM dose-dependently inhibits proliferation (E) and induces apoptosis
(F) of paclitaxel-resistant esophageal cancer cells. ESO26-PAC-r and KYAE-1-PAC-r are resistant to 100 nM and 200 nM paclitaxel, respectively. Resistant cells were maintained in the culture medium containing paclitaxel. Control was set as 1 and shown as dot line. Results were obtained from at least three-independent experiments with triplicate. *p<0.05, compared to control. #p<0.05, compared to paclitaxel.

Fig. 3: Niclosamide acts on paclitaxel-resistant esophageal cancer cells via inhibiting Wnt/β- catenin, STAT3 and mTORC1 pathways. Representative western blot photo (A) and quantification of western blot bands using Image J (B) show decreased levels of Dvl2, β-catenin, p-STAT3 (Tyr705) and p-S6K (Thr389) in paclitaxel-resistant esophageal cancer cells. Cells were treated with niclosamide for 24 h prior to WB analyses. (C) Niclosamide inhibits TOPflash activation in paclitaxel-resistant esophageal cancer cells. Cells transfected with TOPflash plasmid were treated as indicated. RLU, relative light units. (D) Representative western blot photo shows decreased levels of Dvl2, β-catenin, p-STAT3 (Tyr705) and p-S6K (Thr389) in paclitaxel-resistant esophageal cancer cells in the absence or presence of LiCl (10 mM). LiCl significantly abolishes the effects of niclosamide in inhibiting proliferation (E) and inducing apoptosis (F) in paclitaxel- resistant esophageal cancer cells. Resistant cells were maintained in the culture medium containing paclitaxel. Results were obtained from at least three-independent experiments with triplicate. *p<0.05, compared to -LiCL.

Fig. 4: Niclosamide overcomes esophageal cancer paclitaxel chemoresistance in vivo. (A) Combination of niclosamide and paclitaxel is significantly more effective in arresting ESO26 tumor growth in mice. Niclosamide (20 mg/kg, once per day, i.p.), paclitaxel (0.5 mg/kg, twice per week, i.p.) and the combination of both were administrated to mice for 3 weeks. (B) Niclosamide significantly inhibits ESO26-PAC-r tumor growth in mice. 30 mg/kg niclosamide was given to mice once per day via intraperitoneal injection.Tegatrabetan *p<0.05, compared to control or paclitaxel.