Mebendazole inhibits tumor growth and prevents lung metastasis in models of advanced thyroid cancer

in Endocrine-Related Cancer
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  • 1 Departments of Neurosurgery and Oncology, Johns Hopkins University, Baltimore, Maryland, USA
  • 2 Genetic Bases of Thyroid Tumors Laboratory, Division of Genetics, Universidade Federal de São Paulo, São Paulo, Brazil

Correspondence should be addressed to J M Cerutti or G J Riggins: j.cerutti@unifesp.br or griggin1@jhmi.edu

*(T Williamson and T B Mendes contributed equally to this work)

The most common thyroid malignancy is papillary thyroid cancer. While a majority respond to therapy and have a favorable prognosis, some papillary thyroid cancers persist. This subset may dedifferentiate to anaplastic thyroid cancer, an aggressive, highly invasive and rapidly fatal cancer. Thyroid cancer patients at risk for disease progression and metastasis need earlier, safer and more effective therapies. The purpose of this translational study was to determine if mebendazole could be repurposed to effectively treat thyroid cancer, in particular before metastasis. In vitro, mebendazole potently inhibited the growth of a panel of human papillary and anaplastic thyroid cancer cells. In papillary (B-CPAP) and anaplastic (8505c) cell lines, mebendazole increased the percentage of cells in G2/M cell cycle arrest and induced late stage apoptosis by activation of the caspase-3 pathway. In aggressive 8505c cells, mebendazole significantly repressed migratory and invasive potential in a wound healing and transwell invasion assay and inhibited expression of phosphorylated Akt and Stat3 and reduced Gli1. In vivo, mebendazole treatment resulted in significant orthotopic thyroid tumor regression (B-CPAP) and growth arrest (8505c), with treated tumors displaying reduced expression of the proliferation maker KI67 and less vascular epithelium as indicated by CD31+ immunohistochemistry. Most importantly, daily oral mebendazole prevented established thyroid tumors from metastasizing to the lung. Given the low toxicity and published anticancer mechanisms of mebendazole, this novel preclinical study of mebendazole in thyroid cancer has promising therapeutic implications for patients with treatment refractory papillary or anaplastic thyroid cancer.

Abstract

The most common thyroid malignancy is papillary thyroid cancer. While a majority respond to therapy and have a favorable prognosis, some papillary thyroid cancers persist. This subset may dedifferentiate to anaplastic thyroid cancer, an aggressive, highly invasive and rapidly fatal cancer. Thyroid cancer patients at risk for disease progression and metastasis need earlier, safer and more effective therapies. The purpose of this translational study was to determine if mebendazole could be repurposed to effectively treat thyroid cancer, in particular before metastasis. In vitro, mebendazole potently inhibited the growth of a panel of human papillary and anaplastic thyroid cancer cells. In papillary (B-CPAP) and anaplastic (8505c) cell lines, mebendazole increased the percentage of cells in G2/M cell cycle arrest and induced late stage apoptosis by activation of the caspase-3 pathway. In aggressive 8505c cells, mebendazole significantly repressed migratory and invasive potential in a wound healing and transwell invasion assay and inhibited expression of phosphorylated Akt and Stat3 and reduced Gli1. In vivo, mebendazole treatment resulted in significant orthotopic thyroid tumor regression (B-CPAP) and growth arrest (8505c), with treated tumors displaying reduced expression of the proliferation maker KI67 and less vascular epithelium as indicated by CD31+ immunohistochemistry. Most importantly, daily oral mebendazole prevented established thyroid tumors from metastasizing to the lung. Given the low toxicity and published anticancer mechanisms of mebendazole, this novel preclinical study of mebendazole in thyroid cancer has promising therapeutic implications for patients with treatment refractory papillary or anaplastic thyroid cancer.

Introduction

The major types of follicular cell–derived thyroid carcinomas are well-differentiated (papillary and follicular), poorly differentiated and undiferentiated (anaplastic) thyroid carcinoma – which vary considerably in their clinical outcome (Haddad et al. 2015). Papillary thyroid cancer (PTC) represents nearly 80% of cases, responds well to conventional treatment and in most cases carries an excellent prognosis (Melck et al. 2010). However, a rising number of PTC patients will suffer from recurrent or persistent disease (Bates et al. 2018). According to analysis of Surveillance, Epidemiology, and End Results (SEER) cancer registry data from 1980 to 2005, there has been a substantial increase in the incidence of advanced-stage papillary thyroid cancers (>5 cm in diameter), resulting in increased annual mortality rates in thyroid cancer in the United States (Lim et al. 2017).

PTC can progress to advanced poorly differentiated (PDTC) or anaplastic (ATC) subtypes without the proper treatment and these subtypes harbor a far worse prognosis (O’Neill & Shaha 2013). PDTC has an intermediate aggressiveness course between PTC and ATC. ATC is fast growing and one of the most lethal of all human cancers that represents ~2% of all thyroid cancers (Ragazzi et al. 2014). Given the rapid course of the disease and poor treatment outcomes, ATC is responsible for nearly half of all thyroid cancer deaths with a median survival of about 5 months and a 1-year survival rate of 20% (Keutgen et al. 2015). From its onset, ATC displays highly invasive behavior with a majority of patients exhibiting regional or distant metastases in the course of their disease, primarily to the lungs (Haddad et al. 2015). ATC is largely resistant to standard chemotherapy with most patients succumbing to uncontrolled local disease, metastatic disease or treatment-related toxicity (De Crevoisier et al. 2004, Nagaiah et al. 2011, Perri et al. 2011).

It has been suggested that molecular classification may explain the underlying characteristics observed in PTC, PDTC and ATC. The genomic landscape of PTC, recently reported by Cancer Genome Atlas Research Network (2014), showed that PTC is a tumor with one of the lowest tumor mutational burdens, and usually harbors only a single driver gene alteration. The author not only confirmed the significance of BRAFV600E or RAS mutations in the pathogenesis of PTC but also showed distinct signaling and transcriptome consequences in the two main molecular subtypes proposed. BRAFV600E-like tumors showed a higher MAPK transcriptional output and lower expression of thyroid genes involved in idodine uptake and metabolism, as compared to that observed in RAS-like PTC (Cancer Genome Atlas Research Network 2014). These findings partially explain the more aggressive phenotype and increased patient mortality in BRAFV600E-positive PTC (Xing 2013).

The genome and transcriptome analysis of PDTC and ATC confirmed that PDTC and ATC arise from differentiated PTC through the accumulation of key additional genetic activation mutations in TERT, EIF1AX, and PI3K pathway genes and inactivation of tumor suppressor genes TP53, NF2 and RB (Landa et al. 2013, Song & Park 2019, Yoo et al. 2019). Although BRAF and RAS were the predominant drivers in PDTC and ATC, TERT promoter mutations and TP53 were highly prevalence in advanced thyroid cancers harboring BRAF or RAS (Landa et al. 2013, Liu et al. 2013, Liu & Xing 2016). The coexisting BRAFV600E and TERT promoter mutations have a synergistic impact on the aggressiveness of thyroid carcinomas, including increased tumor recurrence and patient mortality (Landa et al. 2016, Liu et al. 2016).

The relationship between the BRAFV600E mutation, increased aggressiveness and poor responsiveness to standard therapies has been partly attributed to higher expression of vascular endothelial growth factor (VEGF) (Jo et al. 2006, Elisei et al. 2008). Thyroid tumors are highly vascularized and rely on VEGF/VEGFR2 signaling for adequate angiogenesis, extrathyroidal invasion, development of metastasis as well as activation of downstream effector pathways such as AKT, BRAF and STAT3 (Viglietto et al. 1995, Keefe et al. 2010, Perri et al. 2011). Therefore, several inhibitors of VEGF signaling pathways have been tested in radioactive iodine-refractory metastatic differentiated thyroid tumors and ATC (Cohen et al. 2008, Sherman et al. 2008). The aggressive pathology of ATC may be further explained by the presence of self-renewing cancer stem cells (Yun et al. 2014); a small population of cells in the tumor that can promote tumor chemo- and radioresistance and lead to high rates of recurrence and metastasis (Vicari et al. 2016). In addition, overactivation of sonic hedgehog (Shh) signaling promotes ATC motility and invasiveness (Williamson et al. 2016a).

Mebendazole (MBZ) is a FDA-approved benzimidazole with an excellent safety profile that has been widely used to treat parasitic infections. The anthelmintic action of MBZ is primarily attributed to its ability to bind tubulin and inhibit microtubule polymerization, an effect that has also correlated with anti-proliferative activity against cancer cells, in vitro (Mukhopadhyay et al. 2002, Bai et al. 2011). MBZ has been repositioned for use in oncology (Pantziarka et al. 2014) because it exhibits preclinical anticancer activity, not only through its known molecular target of tubulin, but also through its off-target ability to bind and inhibit signaling of select tyrosine kinases: VEGFR2, TNIK and BRAF (Nygren et al. 2013, Bai et al. 2015, Tan et al. 2016, Simbulan-Rosenthal et al. 2017). Other reported tumor suppressive mechanisms of MBZ include induction of cell cycle arrest, inhibition of tumor neovascularization, inhibition of the Shh pathway and activation of apoptosis through BCL-2 and caspase-3-dependent mechanisms (Sasaki et al. 2002, Doudican et al. 2008, 2013, Bai et al. 2015, Larsen et al. 2015, Pinto et al. 2015).

MBZ appears to work by targeting multiple oncogenic pathways in cancer cells without the risk of serious side effects (Mudduluru et al. 2016). Evidence supports the use of MBZ for treatment of brain and colon cancers, both in a therapeutic and preventative context, respectively (Bai et al. 2011, 2015, Williamson et al. 2016b). Clinical trials using MBZ in glioblastoma, recurrent pediatric brain tumors and colon cancer are currently under­way (NCT01729260, NCT02644291, NCT03628079). Additionally, clinical case studies of MBZ usage resulted in disease stabilization and tumor regression in refractory metastatic colorectal cancer and metastatic adrenocortical carcinoma (Dobrosotskaya et al. 2011, Nygren & Larsson 2014). We investigated the utility of MBZ as a therapy to treat advanced or refractory thyroid cancers.

In this translational study, we determined MBZ drug sensitivity and effect on in vitro cell proliferation in a panel of human PTC (B-CPAP and TPC1) and ATC (8505c and KTC-2) cell lines. B-CPAP and 8505c cell lines were subjected to further investigation to analyze the effect of MBZ on cell cyle arrest and apoptosis. Aggressive 8505c cells were treated with MBZ and the resulting effect on metastatic potential and select pathways that drive thyroid tumor progression were determined. We then investigated the in vivo effects of daily MBZ treatment on thyroid tumor growth and pulmonary metastasis in orthotopic models of B-CPAP as well as 8505c and analyzed treated tumors for markers of proliferation and angiogenesis.

Materials and methods

Cell culture

8505c (ACC 219) and B-CPAP (ACC 273) cell lines were purchased from DSMZ (Germany). TPC1 cell line was donated by Dr Joao R Maciel (Federal University São Paulo, São Paulo, Brazil). KTC-2 cell line was donated by Prof. Junichi Kurebayashi (Kawasaki Medical School, Japan) (Kurebayashi et al. 2003). Short tandem repeat (STR) sequencing of cell lines was performed to check for cross contamination (Saiselet et al. 2012, Schweppe 2012). B-CPAP, TPC1 and 8505c cells were grown in RPMI-1640 medium supplemented plus 10% FBS. KTC-2 cells were grown in RPMI-1640 medium supplemented plus 5% FBS. Cells were maintained at 37°C in a 5% CO2 humidified atmosphere.

Mebendazole IC50, proliferation

To obtain the half-maximal inhibitory concentrations (IC50) values, B-CPAP, TPC1, 8505c and KTC-2 cells were seeded in 96-well plates (2500/well), cultured for 24 h, then exposed to a range of MBZ concentrations (1 nM - 100 µM) and 1% DMSO (vehicle control). The absorbance of the cells was measured after 48 h and the MBZ IC50 was calculated using GraphPad Prism 5 software. To measure proliferation, cells were seeded (103) in 96-well plates cultured for 24 h before being exposed to MBZ (0.5 µM) and DMSO (control). The proliferation rate was measured by estimating cell viability every 24 h up to 96 h. The number of live cells present in each assay listed above was estimated by using the Cell Counting Kit-8 (CCK8) Assay (Dojindo Laboratories) and measured at an absorbance of 450 nm on a PerkinElmer VICTOR3 plate reader, according to the manufacture’s instructions, as previously described (Bai et al. 2011).

Cell cycle analysis

Cell cycle analysis was performed using propidium iodide staining. B-CPAP and 8505c cells were cultured and treated with 0.5 µM, 1 µM MBZ or vehicle control for 24 h, washed with PBS and fixed in 70% ethanol overnight at 4°C. Cells were pelleted, washed twice with PBS, and stained with 5 µg/mL RNase and 50 µg/mL of propidium iodide (Abcam) in the dark at 37°C for 30 min. The distribution of cells in sub-G1, G1, S and G2/M phases and cells in the 8 N population were determined by flow cytometry on a LSRFortessa cell analyzer (BD Biosciences) and data analysis was performed using the Watson pragmatic model in FlowJo software v10.5.3.

Apoptosis assay

Measurements of viable, early apoptotic and late apoptotic cells were performed on B-CPAP and 8505c cells exposed to 0.5 µM, 1 µM MBZ or vehicle control for 48 h in culture. Cells were detached with Accutase, washed twice with PBS and resuspended in binding buffer and double stained with 5  μL of APC-conjugated Annexin V and 5 μL 7-amino-actinomycin D (7-AAD), per manufacturer’s instructions (BD Pharmigen). Cells were collected by flow cytometry on a BD LSRFortessa cell analyzer and the percentage of cells undergoing apoptosis was determined using FlowJo single-cell analysis software v10.5.3.

Western blotting

Cell homogenates prepared from B-CPAP and 8505c cell lines were incubated on ice in radio-immunoprecipitation (RIPA) buffer supplemented with protease and phosphatase inhibitor cocktails and centrifuged at 10,000 g for 10 min. Protein concentration was quantified using the BCA protein assay kit (Pierce Biotechnology). Thirty micrograms of reduced protein was loaded into a 4–12% NuPage Bis-Tris gel (Invitrogen) and subsequently transferred to a PVDF membrane (BioRad) and blocked for 1 h in TBS with 0.1% Tween/5% nonfat dry milk prior to probing with the following primary antibodies: XIAP, caspase 3, cleaved-caspase 3 (CI-Caspase 3), PARP, cleaved PARP (CI-PARP), total ERK, p-ERK (Thr202/204), total Akt, p-Akt (Ser473), total STAT3, p-STAT3 (Y705), Gli1 and GAPDH (Cell Signaling Technologies). Primary antibodies were incubated overnight at 4°C in blocking buffer then washed and incubated with anti-rabbit horseradish peroxidase-conjugated secondary antibodies for 1 h prior to detection. Signals were visualized using enhanced chemiluminescent reagents according to manufactures instructions (SuperSignal, Thermo Scientific).

Wound healing

8505c cells were cultured in 100 mm culture dishes until they reached near confluence. Multiple linear scratches of equal width were introduced through the confluent cells using a 100 μL pipette. Cells were washed to remove debris and then exposed to 0.5  μM MBZ or vehicle control. A photomicrograph was taken at five locations in each dish at 0 h and 24 h to compare differences in cell migration. The distance of cell migration was measured for each condition and percent relative wound closure rate was estimated to determine the effect of MBZ on cell mobility.

Transwell invasion assay

Transwell invasion assays were performed using a Biocoat matrigel matrix (BD Biosciences) 8-μm pore size, PET membrane in a 24-well plate, according to the manufacturer’s instructions (Corning). The lower chambers of the transwell plates were filled with 750 μL RPMI 1640 medium containing 10% FBS. Following detachment of 8505c cells from tissue culture plates, cells were resuspended in RPMI 1640 medium containing 1% FBS and then loaded onto the upper side of the chamber (2.5 × 104 cells/well in 500 µL media). Transwells were placed in incubators at 37°C for 24 h. The cells on the upper surface of filters were removed using cotton swabs and those on the lower surface were stained with 0.1% crystal violet and counted. Cells that invaded were counted in five random fields of each filter under a microscope.

Luciferase expression

A lentiviral construct with firefly luciferase cDNA was transfected into B-CPAP and 8505c cell lines, with 8 mg/mL polybrene (Sigma Aldrich) in RPMI 1640 media. Cells were maintained at 37°C in a 5% CO2 humidified atmosphere. After 24 h, the cells were harvested and the presence of luminescence was confirmed on a VICTOR3 plate reader. Luciferase-positive cells were further expanded for use in the orthopic thyroid model.

Orthotopic study

All animal experiments were performed according to approved Johns Hopkins Animal Care and Use protocols. For orthopic thyroid implantation, 10-week-old female athymic nude mice were anesthetized using an intraperitoneal injection of ketamine hydrochloride (100 mg/mL) and xylazine (100 mg/mL) diluted in a 10:1 ratio with 0.9% saline. Thyroid cell implantation was performed as previously described (Nucera et al. 2009). Briefy, the neck was washed with betadine and a 1 mm vertical incision was made through the skin and s.c. tissues. The strap muscles were bluntly dissected away to expose the trachea and thyroid glands. One million luciferase-expressing cells were resuspended in 10 µL serum-free media implanted into the right thyroid gland using a Hamilton Syringe (B-CPAP, n = 12 mice) and (8505c, n = 12 mice). Following the implantation, the neck muscles were repositioned and the incision was closed using three nylon sutures. Five days after implantation, thyroid tumor establishment was confirmed by luminescence and the mice were randomized into Control and MBZ, n = 6 mice per group, with equivalent baseline radiance values. Both control and treatment groups were given standard mouse meal with added water and seasame oil (at a 4:1 ratio) necessary for mixing. Seasame oil was supplemented to aid in the absorption of MBZ (Bai et al. 2011). The control mice received untreated feed and the MBZ mice received feed containing 50 mg/kg of mebendazole. Daily MBZ treatment continued starting on Day 5 post-implantation and continued for the duration of the study. Fresh food was prepared 3× per week and daily consumption was calculated to confirm drug dose.

In vivo and ex vivo luciferase activity

Intrathyroidal luciferase activity was measured weekly during the course of the study by injecting the mice with 150 µL (2 µg/mouse) of D-luciferin potassium salt (Gold Bio) in PBS. Mice were then briefly anesthetized with Flurido (Isoflurane, USP) and bioluminescence values were acquired using a Xenogen (IVIS 200, equipped with Living Image Software) within 10 min of luciferin injection, as described previously (Bai et al. 2011). All tumor bioluminescence values were expressed as radiance (photons/second) and graphed to monitor tumor growth in vivo. At the conclusion of the study, the last in vivo measurement was taken and the mice were killed and the thyroid tumor and lungs were quickly harvested. The lungs were immediately placed back in the Xenogen IVIS and exposed for 5 min to visualize ex vivo luminescence for detection of metastasis.

Histology and immunohistochemistry

Thyroid and lung tissues from orthotopic study were placed in 10% formalin at the time of necropsy for subsequent histological analysis. Hematoxylin and eosin (H&E) slides were prepared from experimental tissue and scanned at 20× using a Aperio AT2 slide scanner. Thyroid tumor size was estimated by measuring length and width of tumors from 20× scanned images of H&E sections using the ImageScope software and tumor size was expressed in square millimeters (L × W = mm2). For immunohistochemistry, paraffin-embedded thyroid tumor slides were deparaffinized with xylene and hydrated through a graded alcohol series. Endogenous peroxidase activity was blocked with 3% hydrogen peroxide (Fisher Scientific) for 10 min. Antigen retrieval was performed using Antigen Retrieval Citra Solution pH 6 (BioGenex) in heat cooker for 30 min, followed by avidin/biotin block (Life Technologies) with 10% goat serum + 1% BSA in PBS and incubated for 30 min. Primary antibody Ki67 (1:500, Abcam) and CD31 (1:50, Thermo Fisher) were incubated overnight. Immunostaining was performed using Super SensitiveTM IHC Detection Systems (BioGenex). Slides were counterstained with hematoxylin and analyzed using a light microscope and scanned at 20× using an Aperio AT2 slide scanner. Ki67 staining intensity was measured using ImageJ and the values for three images from n = 5 animals per group were averaged. To analyze microvessel density (MVD), tumor sections were viewed under 20× magnification to identify microvessel ‘hotspots’ of CD31-positive endothelial cells and the average MVD was calculated from ten fields from each treatment group.

Statistical analysis

The results were subjected to Shapiro–Wilk Normality Test. Student’s t-test was used for normally distributed data and Mann–Whitney for data not normally distributed. Values were considered significantly different with a P < 0.05. We used GraphPad Prism 5 (GraphPad Software) and SigmaPlot 12.0 (Systat Software Inc) for statistics.

Results

Mebendazole reduced proliferation and viability of human thyroid cancer cell lines

MBZ IC50 values for human papillary thyroid cell lines B-CPAP and TPC1 were 57 nM and 327 nM, respectively (Fig. 1A and B). For the 8505c and KTC-2 anaplastic thyroid cancer cell lines, the MBZ IC50 values were 472 nM and 146 nM, respectively (Fig. 1C and D). Cell proliferation measured at multiple time points using CCK8 assay revealed that MBZ (0.5 µM) caused significant and sustained cell growth inhibition of 89 and 69% in B-CPAP and TPC1 papillary cell lines, respectively (Fig. 1E and F) and an inhibition of 89 and 72% in 8505c and KTC-2 anaplastic cell lines, respectively (Fig. 1G and H), compared to controls (Fig. 1). These results are consistent with previously published data showing that MBZ can elicit anti-proliferative effects in vitro in a variety of cancer cell lines at concentrations ranging from 0.1 µM to 0.8 µM, regardless of mutation (Mukhopadhyay et al. 2002, Williamson et al. 2016b). The cell lines and their respective MBZ IC50 values and driver mutations are summarized in Fig. 1I.

Figure 1
Figure 1

Mebendazole reduced cell proliferation in papillary and anaplastic thyroid cancer cell lines. The half-maximal inhibitory concentration (IC50) of MBZ in (A) B-CPAP, (B) TPC1, (C) 8505c and (D) KTC-2. Cell proliferation was measured at multiple time points using CCK8 assay up until 96 h after exposure to 0.5 µM MBZ in (E) B-CPAP, (F) TPC1, (G) 8505c and (H) KTC-2 cells. Summary of cell line comparison (I) Data respresent mean ± s.d. P values *<0.05, **<0.01, ***<0.0001.

Citation: Endocrine-Related Cancer 27, 3; 10.1530/ERC-19-0341

Mebendazole induced G2/M cell cycle arrest and caspase 3-dependent apoptosis in thyroid cancer cells

We next investigated whether MBZ induces cell cycle arrest in B-CPAP and 8505c cell lines using propidium iodide staining. At 0.5 µM, MBZ increased the percentage of cells in G2/M phase mitotic arrest by 8.9% in B-CPAP (P = 0.0036) and 1% in 8505c (ns) versus vehicle control-treated cells. At 1 µM, MBZ significantly increased the percentage of cells in G2/M phase mitotic arrest by 44.9% in B-CPAP (P < 0.0001) and 9.9% in 8505c (P = 0.0204) compared to vehicle control cells (Fig. 2A and B). MBZ was able to disrupt cell division in both thyroid cancer cell lines, with B-CPAP cells displaying enhanced sensitivity. The number of 8505c cells in the 8 N population increased in a dose-dependent manner when treated with MBZ which is likely due to mitotic slippage caused by microtubule inhibition.

Figure 2
Figure 2

Mebendazole induces G2/M cell cycle arrest in human papillary and anaplastic thyroid cancer cell lines. (A) Representative histogram plots of cell cycle alterations in B-CPAP and 8505c cells treated with 0.5 µM and 1 µM MBZ or DMSO vehicle control for 24 h. (B) The percentage of cells in each phase of cell cycle at 0.5 µM and 1 µM MBZ were graphed. Data represent mean ± s.d. P values *<0.05, **<0.01, ***<0.0001.

Citation: Endocrine-Related Cancer 27, 3; 10.1530/ERC-19-0341

To compare rates of apoptosis, B-CPAP and 8505c cells were treated with MBZ for 48 h, stained with Annexin V/7-AAD and analyzed by flow cytometry. Figure 3A shows representative dot plots displaying the percentage of cells undergoing early and late stage apoptosis for vehicle control, 0.5 µM MBZ and 1 µM MBZ-treated cells. Compared to controls, 0.5 µM MBZ resulted in 17 and 0.8% increases in the percentage late apoptotic cells in B-CPAP (P = 0.0009) and 8505c (ns), respectively. Compared to controls, 1 µM MBZ resulted in 40.8 and 6.4% increases in the percentage late apoptotic cells in B-CPAP (P = 0.0003) and 8505c (P = 0.0239), respectively (Fig. 3B). As MBZ has been previously shown to selectively induce tumor cell apoptosis via caspase-dependent mechanisms (Sasaki et al. 2002, Doudican et al. 2013, Skibinski et al. 2018), treated cells were probed for markers of apoptosis. Expression of XIAP, a negative regulator of caspase activation, was reduced in B-CPAP cells following MBZ treatment, resulting in significant upregulation of cleaved (activated) caspase 3 and cleaved PARP, even at low (0.25 µM) MBZ concentrations. XIAP expression remained unchanged in MBZ treated 8505c cells and changes in expression of cleaved-caspase and cleaved-PARP followed a more dose-dependent pattern (Fig. 3C). Our in vitro data indicate that B-CPAP is sensitive to MBZ-induced apoptosis, while 8505c cells exhibited greater resistance to apoptosis at lower MBZ concentrations. However, MBZ did induce apoptosis more effectively in 8505c cells than the commonly used BRAF inhibitor, vemurafenib (PLX4032), as indicated by increased expression of cleaved-PARP following 24 h and 6 days of exposure at equivalent drug concentrations (0.5 µM and 1 µM) (Fig. 3D).

Figure 3
Figure 3

MBZ induces caspase 3-dependent apoptosis in aggressive thyroid cancer cells. B-CPAP and 8505c cells were treated with 0.5 µM and 1 µM MBZ or DMSO vehicle control for 48 h and apoptosis was analyzed by flow cytometry using APC Annexin V/7-AAD. (A) Representative dot plot of apoptosis assay by flow cytometry. (B) The percentage of cells undergoing early and late apoptosis at each MBZ concentration were graphed. (C) Expression of proteins involved in MBZ-induced apoptosis were analyzed by Western blot, with GAPDH as the loading control. (D) Expression of PARP/cleaved-PARP was compared in 8505c cells treated with Mebendazole (MBZ) or Vemurafenib (VEM) for 24 h and 6 days to compare activation of apoptosis. Data represent mean ± s.d. P values *<0.05, **<0.01, ***<0.0001. A full colour version of this figure is available at https://doi.org/10.1530/ERC-19-0341.

Citation: Endocrine-Related Cancer 27, 3; 10.1530/ERC-19-0341

Mebendazole suppresses metastatic potential of 8505c and reduces PI3K/Akt and STAT3 signaling

To investigate whether MBZ could inhibit invasive potential of thyroid cancer cell lines, we utilized highly metastatic 8505c cells in in vitro assays of migration and invasion. 8505c cells were exposed to MBZ (0.5 µM) for 24 h and the relative wound closure rate was compared to that of vehicle control treated cells. MBZ significantly reduced 8505c cell migration potential compared to control (P < 0.0001) (Fig. 4A and C). Furthermore, 0.5 µM and 1 µM MBZ significantly inhibited the number of cells that migrated through a Matrigel membrane over 24 h compared to control cells (P < 0.0001) (Fig. 4B and D). MBZ-treated 8505c cells were probed for effectors of the MAPK, PI3K–AKT, STAT3 and Hedgehog signaling pathways. MBZ reduced expression of p-Akt (Ser473), p-Stat3 (Y705) and Gli1, but did not alter expression of p-ERK (Thr202/204) (Fig. 4E).

Figure 4
Figure 4

Mebendazole suppresses metastatic potential of anaplastic 8505c cells and inhibits p-Akt, p-STAT3 and Gli1 expression. Wound healing and transwell assay were used to test the ability of MBZ to inhibit migration and invasion of 8505c cells after 24 h of treatment. (A) Representative photomicrograph of wound healing. (B) Representative photomicrograph of transwell assay. (C) Analysis of relative wound closure of the cells after treatment with MBZ or Control. (D) Analysis of average cells per field in the each group. (E) Representative blot of ERK (total and p-Thr202/204), AKT (total and p-Ser473), STAT3 (total and p-Y705) and Gli1 determined by Western blot with GAPDH as the loading control. Data represent mean ± s.d. ***P < 0.0001.

Citation: Endocrine-Related Cancer 27, 3; 10.1530/ERC-19-0341

Mebendazole inhibits tumor growth and blocks lung metastases in orthotopic models of papillary and anaplastic thyroid cancer

Orthotopic models of thyroid cancer are commonly used to test systemic therapies as they faithfully recapitulate the clinical presentation of rapid tumor growth, tracheal and esophageal compression, laryngeal and tracheal invasion, cachexia, and high incidence of local and distant metastasis seen in the human disease (Kim et al. 2005, Antonello & Nucera 2014). Orthotopic xenografts were established in athymic nude mice using luciferase-expressing B-CPAP and 8505c cells. Bioluminescent images from control and MBZ-treated mice were acquired at weekly intervals over the course of the study to monitor tumor growth (Figs 5A and 6A). Following a 5-day establishment period, mice were randomized and given either control or daily 50 mg/kg MBZ in the feed for 42 days (B-CPAP) and 28 days (8505c) and the experiment was terminated when control mice began exhibiting signs of cachexia.

Figure 5
Figure 5

Mebendazole inhibits primary tumor growth and metastasis to lungs in orthotopic model of papillary thyroid cancer. (A) One million luciferase expressing B-CPAP cells were implanted into the right thyroid of nude mice and in vivo bioluminescence was obtained weekly to monitor tumor growth between control and 50 mg/kg MBZ-treated mice. (B) Thyroid intratumoral luminescence was graphed during 42 days of treatment. (C) At the end of the study, thyroid and surrounding tissue were extracted for histological analysis revealing large tumors in the Control group (T) and small microtumors in MBZ group (indicated by arrow). (D) The lungs were removed for ex vivo bioluminescent imaging of metastasis and (E) histological evaluation of pulmonary metastatic lesions. Data represents mean ± s.d. P values *<0.05, **<0.01.

Citation: Endocrine-Related Cancer 27, 3; 10.1530/ERC-19-0341

Figure 6
Figure 6

Mebendazole inhibits primary tumor growth and metastasis to lung in orthotopic model of anaplastic thyroid cancer. (A) One million luciferase expressing 8505c cells were implanted into the right thyroid of nude mice and in vivo bioluminescence was obtained weekly to monitor tumor growth between control and 50 mg/kg MBZ-treated mice. (B) Thyroid intratumoral luminescence was graphed during 28 days of treatment. (C) At the end of the study, the thyroid and surrounding tissue were extracted for histological analysis revealing large, infiltrating tumors in the Control group (T) compared to smaller tumors and normal left thyroid (N) in the MBZ group. (D) The lungs were removed for ex vivo bioluminescent imaging of metastasis and (E) histological evaluation of pulmonary metastatic lesions. Data represent mean ± s.d. P values *<0.05, **<0.01.

Citation: Endocrine-Related Cancer 27, 3; 10.1530/ERC-19-0341

In the B-CPAP papillary thyroid cancer model, the tumors of the untreated mice displayed a significant increase in radiance 20 days post implantation and the tumors continued to grow steadily, with a sharp rise in tumor radiance between days 40–49 post implantation (Fig. 5B). In contrast, the imaging of MBZ-treated B-CPAP mice showed a marked reduction in radiance after 14 days of treatment and the radiance was completely undetectable in four mice and nearly undectectable in two mice at the end of the study (Fig. 5A). Overall, MBZ treatment of B-CPAP orthotopic tumors resulted in a 99% (P = 0.0072) radiance decrease compared to control (Fig. 5B). Thyroid tumors were palpable in 83% (5/6) untreated B-CPAP mice and H&E sections revealed massive primary tumors (median size 45.5 mm2) that were invading locally into the surrounding tissue (Fig. 5C). In contrast, MBZ-treated B-CPAP mice did not have palpable tumors and H&E sections revealed small, microscopic tumors embedded in the thyroid (median size 0.041 mm2) or no tumor (Fig. 5C). The left thyroid and surrounding tissues were unaffected in MBZ-treated mice. Ex vivo analysis of lung tissue revealed bioluminescent metastatic lesions in 83% (5/6) of the mice in the B-CPAP control group, whereas there was no detectable luminescence in the lungs of MBZ-treated mice (Fig. 5D). H&E-stained lung tissue sections were used to verify the presence of multiple micrometastic lesions in the control group and unaffected lung tissue in MBZ-treated mice (Fig. 5E).

In the 8505c, orthotopic model of anaplastic thyroid cancer, weekly in vivo bioluminescent images obtained over the course of the study (Fig. 6A) showed the rapid course of the disease as thyroid tumors in untreated mice grew in a predictable, aggressive manner (Antonello & Nucera 2014, Morrison et al. 2015). Radiance values from 8505c control mice began to significantly increase 20 days post implantation, followed by a sharp growth period between day 28 and 35 post implantation (Fig. 6B). It was during this time that the control mice presented with evidence of the mass effect of the tumor on the trachea and invasion to the lungs. All 100% (6/6) of mice in the control group had large, palpable tumors on day 35. In the MBZ group, the treatment inhibited tumor growth and stabilized radiance values, resulting in a 96% (P = 0.0140) reduction in final intratumoral luminescence at 35 days post implantation when compared with control mice (Fig. 6A and B). Analysis of H&E sections confirmed that the control 8505c mice had large, infiltrating primary thyroid tumors (median size 29.9 mm2) that were locally invading the trachea and left thyroid (Fig. 6C). MBZ-treated 8505c mice had small tumors (median size 1.71 mm2) that were confined to the right thyroid only with no indication of local invasion (Fig. 6C). During the ex vivo lung analysis, the presence of pulmonary mestastases was vividly detected via luminescence in 100% (6/6) of mice in the control group, whereas in the MBZ-treated group there was no evidence of metastasis (Fig. 6D). H&E staining of lung tissue allowed visualization of widespread metastasis in the control mice, which was not observed in the MBZ-treated mice (Fig. 6E). The results of our orthotopic mouse studies show that MBZ suppresses thyroid tumor growth and blocks lung metastasis in two aggressive, BRAF, TP53, TERT-mutant models.

Mebendazole decreases markers of tumor proliferation and vascularization in anaplastic thyroid carcinoma

The tumors derived from the 8505c thyroid studies were further analyzed for markers of proliferation (Ki67) and microvessel density (CD31) (Fig. 7A). Immunohistochemical analysis of Ki67 was used to visualize MBZ effects on cell proliferation in vivo, to correlate with induction of mitotic arrest, in vitro (Mukhopadhyay et al. 2002, Sasaki et al. 2002). MBZ decreased Ki67 staining intensity in 8505c orthotopic tumors by 61% (P = 0.0007) compared to untreated controls (Fig. 7B). It has been previously shown that MBZ acts as a kinase inhibitor against VEGFR2 and reduces tumor angiogenesis (Bai et al. 2015, Williamson et al. 2016b). To measure effects on the tumor vasculature, we calculated microvessel density in tumor sections stained with the anti-CD31 (PECAM1) antibody. MBZ significantly decreased CD31-positive endothelial staining in 8505c orthotopic thyroid tumors 47% (P = 0.0202) (Fig. 7C). It should be noted that control B-CPAP thyroid tumors also showed intense Ki67 and CD31 positivity (data not shown) but lack of sufficient tumors in MBZ treated B-CPAP mice did not allow for an evaluation of treatment effect. Therefore, only 8505c tumors were subjected to comparative analysis.

Figure 7
Figure 7

Mebendazole suppressed tumor cell proliferation and vascularization in orthotopic model of anaplastic thyroid cancer. (A) Immunohistochemical staining of H&E, Ki67 and CD31 in 8505c orthotopic thyroid tissue. Treatment with daily 50 mg/kg MBZ suppressed the percentage of Ki67-positive proliferating cells (B) and CD31-positive microvessel density (C) compared to untreated controls. Data represent mean ± s.d. P values *<0.05, ***<0.001.

Citation: Endocrine-Related Cancer 27, 3; 10.1530/ERC-19-0341

Discussion

Our preclinical findings indicate that a non-toxic oral benzimidazole, mebendazole, can suppress primary thyroid tumor growth and prevent pulmonary metastasis. This has important clinical implications for the 20–30% of PTC patients (12,000–18,000 patients in the United States) who are at risk of developing persistent or progressive disease, which could dedifferentiate into a more aggressive undifferentiated phenotype with lower survival rates (Melck et al. 2010, Czarniecka et al. 2016, Bates et al. 2018). There are limited FDA-approved drugs for patients with advanced or recurrent thyroid cancer and the evolving treatment regimens have yet to have a significant impact (Keutgen et al. 2015, Saini et al. 2018). In recent years, there has been extensive clinical testing of targeted kinase inhibitors as second-line therapies (Sherman 2011, Guerra et al. 2013, Hsu et al. 2014, Alonso-Gordoa et al. 2015), but many of these therapies fail due to drug resistance or unacceptable drug toxicity (Viola et al. 2016).

In this study, we demonstrated that oral mebendazole polymorph C can effectively inhibit the growth and viability of thyroid cancer cells at nanomolar concentrations, in vitro. The MBZ IC50 values for papillary thyroid cancer cell lines, B-CPAP and TPC1, were 57 nM and 327 nM, respectively, and for anaplastic thyroid cancer cell lines, 8505c and KTC-2, were 472 nM and 146 nM, respectively. This data indicates a difference in drug sensitivity between the all cell lines, although a strong correlation between IC50 and mutation profile was not observed. B-CPAP cells were highly sensitive to MBZ-induced cell cycle arrest and apoptosis, even at low concentrations. 8505c cells exhibited a dose-dependent increase of apopstosis susceptibility to MBZ. The loss of the tumor suppressor NF2 in 8505c cells is one possible explaination for the increased resistance to apoptosis (Garcia-Rendueles et al. 2015). Importantly, MBZ was more effective at inducing cell death in 8505c cells than the commonly used BRAF-inhibitor, vemurafenib. Vemurafenib inhibits MAPK signaling in thyroid cancer but cells do not undergo apoptosis when treated with BRAFV600E inhibitors, suggesting the persistence of additional signaling pathways that promote survival and drug resistance, such as upregulated PI3K/Akt signaling (Vanden Borre et al. 2014).

The underlying mechanisms of MBZ that resulted in growth and metastasis suppression were further investigated in 8505c cells. We demonstrated that MBZ can suppress the migratory and invasive potential of 8505c cells in a wound healing and transwell invasion assay. This is consistent with the observed decreased expression of both p-Akt (Ser473), p-STAT3 (Y705) and Gli1 in MBZ-treated 8505c cells. This effect could be explained by upstream VEGFR2 kinase inhibition by MBZ. Although the role of abherent PI3K and Hedgehog signaling in thyroid cancer is well documented, the role of STAT3 has been more controversial. One study showed that activated STAT3 is tumor suppressive in PTC and ATC (Couto et al. 2012), while others have demonstrated that activated STAT3 has been shown to be significantly upregulated in PTC tumors compared to paracancerous tissue and that increased p-STAT3 corresponded with higher rates of lymph node metastasis (Zhang et al. 2011, Yan et al. 2015). The role of JAK/STAT3 signaling in promoting expansion of treatment-resistant cancer stem cells in ATC was recently demonstrated (Shiraiwa et al. 2019). Flubendazole, another anthelmintic benzimidazole, has been shown cause dysregulation of STAT3 activation in triple-negative breast cancer cells, resulting in decreased breast cancer cell stemness and mammosphere-forming abilities (Oh et al. 2018). Inhibition of STAT3 in ATC has important therapeutic implications in reducing rates of disease recurrence and increasing responsiveness to immunotherapies (Tseng et al. 2012, Attili et al. 2018). Further studies would be necessary to fully elucidate the suppressive effects of MBZ on thyroid cancer stem cell populations and its potential role in cancer immune-modulation.

In our preclinical study, we observed significant tumor growth inhibition in both B-CPAP and 8505c orthotopic thyroid models using a single, non-toxic drug. The in vitro difference in drug response translated into our in vivo studies, as treatment with MBZ caused significant regression in tumor size in the B-CPAP orthotopic tumors, whereas MBZ arrested the growth of 8505c tumors. It is worth noting that B-CPAP, although classified as a papillary carcinoma, also shares common features of poorly differentiated thyroid carcinoma (Landa et al. 2016). Several preclinical publications using these same models report the sensitivity of B-CPAP to a therapy but note that 8505c is largely resistant to treatment (Chan et al. 2012, Gunda et al. 2014, Vanden Borre et al. 2014). Two and three drug combinations are often needed to suppress 8505c in vivo (Gunda et al. 2014), which may translate into increased adverse drug toxicity in the clinical setting. We observed that MBZ monotherapy constrained 8505c orthotopic tumors sufficiently to prevent distant metastasis to the lung, despite the fact that widespread metastasis was seen in untreated animals. These observations have direct clinical relavance, as distant metastasis to the lungs is common in ATC patients and is a significant cause of disease-related morbidity and mortality.

The main focus of this translational study was to predict the efficacy of mebendazole in the worst thyroid cancer subtypes. These experiments indicate that mebendazole reduced cell proliferation and decreased tumor neo-vascularization in vivo, which is consistent with previous descriptions of the anticancer mechanism of MBZ. Smaller tumors with reduced vasculature would have less capacity to invade beyond the thyroid capsule and into surrounding lymph nodes. MBZ is known to prevent the polymerization of tubulin, the molecular target of the widely used anticancer drugs paclitaxel and vincristine, and induce mitotic arrest selectively in tumor cells without serious adverse side effects (Pinto et al. 2015). ATC and PDTC are aggressive, genomically heterogeneous thyroid cancers with some of the shortest predicted survivals of any cancer. In these cases, a multimodality treatment approach is likely the only way to significantly delay disease progression and increase survival. The use of a safe, non-toxic drug mebendazole as an adjuvant therapy following surgical resection or in combination with radiotherapy, targeted chemotherapy or immunotherapy could increase the durability of treatment responses in ATC, without added toxicity. Repurposed, FDA-approved drugs with known toxicity profiles, such as mebendazole, are more readily adapted to clinical trials with less risk than newly developed drugs. The evidence presented here using preclinical models of aggressive thyroid cancer subtypes, combined with the safety track record of high dose oral mebendazole, suggest a clinical trial against these cancers should be attempted.

Declaration of interest

G J R and T W are inventors on intellectual property related to mebendazole owned and managed by Johns Hopkins University conflict of interest policies. Dr Riggins is a founder of and holds equity in Benizole Therapeutics, PBC. The results of the study discussed in this publication could affect the value of Benizole. This arrangement has been reviewed and approved by the Johns Hopkins University in accordance with its conflict of interest policies. The other authors have nothing to disclose.

Funding

Funding for this study was provided by National Institutes of Health grants U01 OH10984 and U01 OH011849-01, the Virginia and D.K. Ludwig Fund, and the São Paulo Reseach Foundation (FAPESP), grant #2014/06570-6 to J M C. T B M is a recipient of a FAPESP fellowship and J M C is a recipient of a Research Productivity Scholarship of from the Brazilian National Council for Scientific and Technological Development (CNPq). G J R is supported by the Irving J. Sherman M.D. Research Professorship.

Author contribution statement

T W helped design and oversee the study, performed experiments in Figs 2, 3 and 4, assisted in mouse studies and histology in Figs 5, 6 and 7 and drafted the paper, T B M performed experiment in Fig. 1, conducted mouse studies and histology in Figs 5, 6 and 7 and helped write the paper, N J performed drug treatment and western blot experiments in Figs 3 and 4, J M C and G J R jointly help design the experiments, reviewed the data, and edited the paper.

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Society for Endocrinology

Sept 2018 onwards Past Year Past 30 Days
Abstract Views 3850 2017 0
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    Mebendazole reduced cell proliferation in papillary and anaplastic thyroid cancer cell lines. The half-maximal inhibitory concentration (IC50) of MBZ in (A) B-CPAP, (B) TPC1, (C) 8505c and (D) KTC-2. Cell proliferation was measured at multiple time points using CCK8 assay up until 96 h after exposure to 0.5 µM MBZ in (E) B-CPAP, (F) TPC1, (G) 8505c and (H) KTC-2 cells. Summary of cell line comparison (I) Data respresent mean ± s.d. P values *<0.05, **<0.01, ***<0.0001.

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    Mebendazole induces G2/M cell cycle arrest in human papillary and anaplastic thyroid cancer cell lines. (A) Representative histogram plots of cell cycle alterations in B-CPAP and 8505c cells treated with 0.5 µM and 1 µM MBZ or DMSO vehicle control for 24 h. (B) The percentage of cells in each phase of cell cycle at 0.5 µM and 1 µM MBZ were graphed. Data represent mean ± s.d. P values *<0.05, **<0.01, ***<0.0001.

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    MBZ induces caspase 3-dependent apoptosis in aggressive thyroid cancer cells. B-CPAP and 8505c cells were treated with 0.5 µM and 1 µM MBZ or DMSO vehicle control for 48 h and apoptosis was analyzed by flow cytometry using APC Annexin V/7-AAD. (A) Representative dot plot of apoptosis assay by flow cytometry. (B) The percentage of cells undergoing early and late apoptosis at each MBZ concentration were graphed. (C) Expression of proteins involved in MBZ-induced apoptosis were analyzed by Western blot, with GAPDH as the loading control. (D) Expression of PARP/cleaved-PARP was compared in 8505c cells treated with Mebendazole (MBZ) or Vemurafenib (VEM) for 24 h and 6 days to compare activation of apoptosis. Data represent mean ± s.d. P values *<0.05, **<0.01, ***<0.0001. A full colour version of this figure is available at https://doi.org/10.1530/ERC-19-0341.

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    Mebendazole suppresses metastatic potential of anaplastic 8505c cells and inhibits p-Akt, p-STAT3 and Gli1 expression. Wound healing and transwell assay were used to test the ability of MBZ to inhibit migration and invasion of 8505c cells after 24 h of treatment. (A) Representative photomicrograph of wound healing. (B) Representative photomicrograph of transwell assay. (C) Analysis of relative wound closure of the cells after treatment with MBZ or Control. (D) Analysis of average cells per field in the each group. (E) Representative blot of ERK (total and p-Thr202/204), AKT (total and p-Ser473), STAT3 (total and p-Y705) and Gli1 determined by Western blot with GAPDH as the loading control. Data represent mean ± s.d. ***P < 0.0001.

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    Mebendazole inhibits primary tumor growth and metastasis to lungs in orthotopic model of papillary thyroid cancer. (A) One million luciferase expressing B-CPAP cells were implanted into the right thyroid of nude mice and in vivo bioluminescence was obtained weekly to monitor tumor growth between control and 50 mg/kg MBZ-treated mice. (B) Thyroid intratumoral luminescence was graphed during 42 days of treatment. (C) At the end of the study, thyroid and surrounding tissue were extracted for histological analysis revealing large tumors in the Control group (T) and small microtumors in MBZ group (indicated by arrow). (D) The lungs were removed for ex vivo bioluminescent imaging of metastasis and (E) histological evaluation of pulmonary metastatic lesions. Data represents mean ± s.d. P values *<0.05, **<0.01.

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    Mebendazole inhibits primary tumor growth and metastasis to lung in orthotopic model of anaplastic thyroid cancer. (A) One million luciferase expressing 8505c cells were implanted into the right thyroid of nude mice and in vivo bioluminescence was obtained weekly to monitor tumor growth between control and 50 mg/kg MBZ-treated mice. (B) Thyroid intratumoral luminescence was graphed during 28 days of treatment. (C) At the end of the study, the thyroid and surrounding tissue were extracted for histological analysis revealing large, infiltrating tumors in the Control group (T) compared to smaller tumors and normal left thyroid (N) in the MBZ group. (D) The lungs were removed for ex vivo bioluminescent imaging of metastasis and (E) histological evaluation of pulmonary metastatic lesions. Data represent mean ± s.d. P values *<0.05, **<0.01.

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    Mebendazole suppressed tumor cell proliferation and vascularization in orthotopic model of anaplastic thyroid cancer. (A) Immunohistochemical staining of H&E, Ki67 and CD31 in 8505c orthotopic thyroid tissue. Treatment with daily 50 mg/kg MBZ suppressed the percentage of Ki67-positive proliferating cells (B) and CD31-positive microvessel density (C) compared to untreated controls. Data represent mean ± s.d. P values *<0.05, ***<0.001.

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