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. 2018 Apr 16;233(9):7391–7401. doi: 10.1002/jcp.26579

Possible repurposing of pyrvinium pamoate for the treatment of mesothelioma: A pre‐clinical assessment

Marcella Barbarino 1,2, Daniele Cesari 1, Riccardo Intruglio 1, Paola Indovina 2, Asadoor Namagerdi 1, Franca Maria Bertolino 1, Maria Bottaro 1, Delaram Rahmani 1, Cristiana Bellan 3, Antonio Giordano 1,2,✉
PMCID: PMC13482138  PMID: 29659015

Abstract

Malignant mesothelioma (MM) is a very aggressive asbestos‐related cancer, whose incidence is increasing worldwide. Unfortunately, no effective therapies are currently available and the prognosis is extremely poor. Recently, the anti‐helminthic drug pyrvinium pamoate has attracted a strong interest for its anti‐cancer activity, which has been demonstrated in many cancer models. Considering the previously established inhibitory effect of pyrvinium pamoate on the Wnt/β‐catenin pathway and given the important role of this pathway in MM, we investigated the potential anti‐tumor activity of this drug in MM cell lines. We observed that pyrvinium pamoate significantly impairs MM cell proliferation, cloning efficiency, migration, and tumor spheroid formation. At the molecular level, our data show that pyrvinium pamoate down‐regulates the expression of β‐catenin and Wnt‐regulates genes. Overall, our study suggests that the repurposing of pyrvinium pamoate for MM treatment could represent a new promising therapeutic approach.

Keywords: mesothelioma, pyrvinium pamoate, Wnt

In this study we challenged malignant mesothelioma (MM) cell lines with pyrvinium pamoate, an anti‐helminthic drug that has proven to have anti‐tumor effects in different cancer models. We observed that this drug significantly impaired MM cell proliferation, clonogenic potential, migration, and tumor spheroid formation. At the molecular level, our data suggest that pyrvinium pamoate cellular effects could, at least in part, be mediated by the inhibition of the Wnt/β‐catenin pathway.

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1. INTRODUCTION

Malignant mesothelioma (MM) is a very aggressive, highly chemo‐resistant solid tumor, arising from the mesothelium covering the serous cavities of the body. Malignant pleural mesothelioma (MPM) is the most common MM type; more rarely MM affects the peritoneum (≈20%), the pericardium and the tunica vaginalis testis (≈1–2%) (Bridda, Padoan, Mencarelli, & Frego, 2007).

Despite the current aggressive multimodal treatment regimen, including chemotherapy, radiation, and surgery, the prognosis remains extremely poor, with a median survival of 9–17 months from diagnosis (Tsao, Wistuba, Roth, & Kindler, 2009).

Asbestos exposure causes the majority of MM cases, with an attributable risk of 80–90% (Lacourt et al., 2014). The association between MPM and asbestos exposure was first described by Wagner, Sleggs, & Marchand, 1960. The extensive use of asbestos during the twentieth century determined an increase in MM incidence and mortality, which are expected to further rise in the coming years, especially for countries where asbestos has not been banned (Abdel‐Rahman, 2018).

Moreover, despite we are today paying for the widespread use of asbestos in the past, mounting evidence is demonstrating that exposure to carbon nanotubes (CNTs) might similarly induce lung cancer and MM (Luanpitpong, Wang, Davidson, Riedel, & Rojanasakul, 2016; Yu et al., 2015). Due to their excellent mechanical, electronic, and optical properties, CNTs, depending on the functionalization type, can be used for many purposes, such as drug delivery, cancer therapy, regenerative medicine, and cell‐ and tissue‐imaging (Erol et al., 2017). Even if with discordant results, this material could have a carcinogenic potential similar to that of asbestos, owing to their biopersistent fibres remaining within the lung for several months (Rom & Palmer, 1974; Vietti, Lison, & van den Brule, 2016). Moreover, the absence of appropriate tools for nanomaterial carcinogenicity testing determines an inappropriate usage of these materials (Kumar & Dhawan, 2013) and leads to the lack of specific guidelines to prevent the associated health risks (Bahadar, Maqbool, Niaz, & Abdollahi, 2016). These observations raise the possibility of a new “manmade tragedy” in the coming years, as was the use of asbestos in the past. Therefore, considering both the expected increase in the incidence of MM and the lack of effective treatment modalities for this cancer type, there is an urgent need to develop new therapeutic approaches.

Pyrvinium pamoate, an anthelminthic drug approved by FDA in the 1950s, has recently attracted particular attention in the field of anti‐cancer research for its anti‐proliferative activity in various cancer models. As recently reviewed by Momtazi‐Borojeni, Abdollahi, Ghasemi, Caraglia, & Sahebkar, 2018, pyrvinium pamoate was demonstrated to act as an anti‐cancer drug inhibiting different molecular oncogenic pathways, depending on the cellular contest. The most studied mechanisms of action include the inhibition of the Wnt signaling pathway (Polosukhina et al., 2017; Wiegering et al., 2014; Xu et al., 2017; Zhang, Zhang, Zhang, Wang, & Hu, 2017), the suppression of mitochondrial respiration (Harada, Ishii, Hatake, & Kasahara, 2013; Ishii, Harada, & Kasahara, 2012; Xiang et al., 2015; Xiao, Zhang, Zhou, Rajoria, & Wang, 2016), the inhibition of the PI3K/AKT/P70S6K pathway (Carrella et al., 2016; Venerando, Girardi, Ruzzene, & Pinna, 2013), the targeting of the androgen receptor (AR)/androgen axis (Jones et al., 2009; Lim et al., 2014) and the suppression of tumor‐sphere formation (Xu et al., 2013). Increasing interest in the role of the Wnt pathway in MM carcinogenesis and resistance to conventional therapies (de Assis & Isoldi, 2014; Fox et al., 2013; He et al., 2005; Perumal, Dharmarajan, & Fox, 2017) prompted us to investigate the potential anti‐tumor activity of pyrvinium pamoate in MM cell lines. In particular, we analyzed the effects of pyrvinium pamoate on cell proliferation, clonogenic potential, migration, and multicellular tumor spheroid formation. Moreover, we also investigated the effects of this drug on some key molecular features of MM biology.

2. MATERIALS AND METHODS

2.1. Mesothelioma cell culture

A panel of MM cells was used in cytotoxicity assays to determine pyrvinium pamoate anti‐proliferative activity. The cell lines utilized were: NCI‐H28, NCI‐H2452 and MSTO‐211H, purchased from the American Type Culture Collection (Rockville, MD); IST‐MES1 and IST‐MES2, obtained from the National Institute of Cancer research, Genova, Italy; REN, kindly provided by Giovanni Gaudino (University of Hawaii Cancer Center, Manoa).

All the cells were cultured in the appropriated medium at 37 °C in a humidified incubator with 5% CO2.

2.2. Cytotoxicity assay

Pyrvinium pamoate was purchased from Sigma–Aldrich. Stock solutions of the drug (0.05 mol/L) were prepared in DMSO (Euroclone) and stored at −20 °C.

Cells were seeded in 96‐well plates (Costar 3599, Corning, NY) 24 hr before treatment with different concentrations of pyrvinium pamoate and incubated for further 72 hr. Control cells were treated with DMSO at the same amount used to deliver the molecule. Each experiment was conducted in triplicate. Cell survival was evaluated by staining with sulforhodamine B (SRB, Sigma–Aldrich) as previously described (Skehan et al., 1990). Absorbance values were measured with a microplate reader (Celbio) at 540 nm. The half maximal inhibitory concentration (IC50) values were calculated using GraphPad Prism 6 (GraphPad Software Inc; http://www.graphpad.com/scientificsoftware/prism/).

2.3. Colony formation assay

MSTO‐211H cells (100/dish) were seeded in 100 mm diameter petri dishes (Euroclone) and 24 hr after seeding were treated with different concentrations of pyrvinium pamoate. After 14 days, during which the medium was refreshed every 2 days, the colonies were fixed with 4% paraformaldehyde for 20 min and stained with crystal violet (Sigma–Aldrich, St. Louis, MO). Cell colonies were photographed and enumerated under an inverted microscope (Zeiss, Germany).

2.4. Protein extraction and western blotting

MM cell lines were seeded in 100 mm diameter dishes and 24 hr after seeding were treated with pyrvinium pamoate at the indicated doses and for the indicated times.

Control cells were treated with DMSO alone. After treatments, the cells were harvested on ice in lysis buffer consisting of 50 mM TRIS‐HCl pH 7.5, 50 mM EDTA pH 8, 150 mM NaCl, 1% NP40, 2 mM NaOV, 10 mM NaF, 0.3 mM PMSF, and a protease inhibitor cocktail (Roche).

Equal amounts of proteins (30 μg) per sample were electrophoresed and blotted onto nitrocellulose membranes (Bio‐Rad, Hercules, CA) and incubated with antibodies against β‐catenin (Life Technology, Carlsbad, CA) and glyceraldehyde‐3‐phosphate dehydrogenase (GAPH, Santa Cruz Biotechnology, Dallas, TX). After incubation with horseradish peroxidase‐conjugated secondary antibodies, signals were detected using the Supersignal West Pico Chemiluminescent Substrate (Pierce, Waltham, MA) and imaged with Bio‐Rad Chemidoc XRS+.

2.5. Real‐time quantitative reverse transcription polymerase chain reaction (qRT‐PCR)

Total RNA from pyrvinium pamoate‐treated MM cells was reverse transcribed with the iScript cDNA Synthesys kit (Bio‐rad). Gene expression was analyzed by real‐time qRT‐PCR using the LightCycler™ instrument (Roche Applied Sciences, Indianapolis, Penzberg, Germany) according to the manufacturer's protocol. The primers used were from Bio‐Rad (Table 1). The housekeeping GAPDH gene was used to normalize the expression of genes of interest. Gene expression levels in treated cells were calculated by the 2−ΔΔCt method (Livak & Schmittgen, 2001) relatively to control cells.

Table 1.

Real‐time qRT‐PCR primers (Bio‐Rad)

Target gene Gene ID
AXIN2 8313
CCND1 59
ROR2 4920
VEGFA 7422
MET 4322
HMGB1 3146
MMP9 4318
GAPDH 2597
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2.6. Wound healing scratch test

Wound healing assays were performed to investigate cell migration in response to treatment. MSTO‐211H cells were grown to confluence in tissue culture dishes (100 mm). A scratch was made in the confluent monolayer using a sterile pipette tip, then the medium was discarded and cells were rinsed twice with PBS. Subsequently, the cells were treated with pyrvinium pamoate or DMSO and incubated at 37 °C. Photographs were taken every 24 hr until the scratch area had closed, using a Zeiss Axiovert microscope (Zeiss, Germany).

2.7. Generation of spheroids

For spheroid inhibition assay, MSTO‐211H cells (1 × 103/well) were seeded in 96 well plates with low cell attachment surface (Thermo Scientific™ Nunclon Sphera™, Waltham, MA), using a sub‐toxic concentration of pyrvinium pamoate (25 nM) or an equal amount of DMSO. Spheroids were observed under an inverted microscope and photographed at 3 and 5 days after seeding. Experiments were performed in triplicate.

2.8. Statistical analysis

Statistical analysis was performed using the GraphPad Prism Software. Statistically significant differences among the means of multiple matched groups were evaluated by one‐way repeated measures Anova with Dunnett post‐test, to compare all data versus control. To evaluate statistically significant differences between the means of two matched groups, we used the two‐tailed paired Student t‐test. p < 0.05 was considered to be statistically significant.

3. RESULTS

3.1. Pyrvinium pamoate suppresses the growth of mesothelioma cell lines

We tested the possible anti‐proliferative activity of pyrvinium pamoate on a panel of MM cell lines. After 72 hr of treatment with this drug at concentrations ranging from 2.5 to 50 nM, we evaluated cell viability by the SRB assay. We observed that pyrvinium pamoate significantly inhibited the proliferation of all the MM cell lines tested at nanomolar concentrations in a dose‐responsive manner (Figure 1). Pyrvinium pamoate IC50 values, calculated 72 hr after treatment, are reported in Table 2.

Figure 1.

Figure 1

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Dose‐response curves of mesothelioma cells treated with pyrvinium pamoate. The cells were treated for 72 hr with pyrvinium pamoate at concentrations ranging from 2.5 to 50 nM. The results are reported as means ± standard deviations of four independent experiments. Data are expressed as percentages of cell viability calculated with respect to control cells treated with DMSO alone and analyzed by nonlinear regression. The absorbance values of treated and control samples were subjected to one‐way Anova with Dunnett post‐test. All drug concentrations gave extremely significant effects with p values <0.001, except the lowest concentrations in NCI‐H28 cells (10 and 40 nM resulted in p values >0.05 and <0.05, respectively) and in IST‐MES1 cells (2.5, 10, and 40 nM resulted in p values >0.05)

Table 2.

IC50 values of pyrvinium pamoate in mesothelioma cell lines

Cell line IC50 (nM)
NCI‐H2452 65.59
REN 56.3
MSTO‐211H 23.58
NCI‐H28 535.6
IST‐MES1 332.3
IST‐MES2 105.7
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Moreover, to evaluate whether this drug exerted a long‐term inhibition of MM cell growth, we performed a clonogenic assay in the most responsive cell line, MSTO‐211H, and observed a significant reduction in colony formation (Figure 2). We focused all our further analyses on this cell line.

Figure 2.

Figure 2

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Long‐term effect of pyrvinium pamoate on MSTO‐211H cell growth, assessed by clonogenic assay. Twenty‐four hours after seeding, MSTO‐211H cells were treated with the indicated concentrations of pyrvinium pamoate for 48 hr and allowed to growth for 14 days, during which the medium was refreshed every 2 days. Control cells (CTR) were treated with DMSO alone. After fixing and staining with crystal violet, the cell colonies were photographed and counted under an inverted microscope. A dose‐response effect of pyrvinium pamoate treatment on MSTO‐211H ability to form colonies was observed

3.2. Pyrvinium pamoate down‐regulates β‐catenin expression in mesothelioma cells

We explored the effects of pyrvinium pamoate on the Wnt/β‐catenin pathway. We firstly analyzed the expression of β‐catenin, the major effector of Wnt signaling transduction (Petrov, Zhidkova, Serikov, Zenin, & Popov, 2012). Indeed, regulation of the level of β‐catenin is the central switch in the Wnt pathway: in the absence of Wnt ligands or inhibiting Wnt stabilization, the levels of β‐catenin decreases due to its degradation by the “destruction complex” consisting of the adenomatous polyposis coli (APC) protein, AXIN, glycogen synthase kinase 3β (GSK3β), and casein kinase 1α (CK1α). In this complex GSK3β and CK1α phosphorylate β‐catenin at residues S33/S37/T41 and S45, respectively, leading to β‐catenin ubiquitination by β‐TrCP (beta‐transducin repeat containing E3 ubiquitin protein ligase) (Hart et al., 1999) and its subsequent degradation. pyrvinium pamoate has been described as a potent inhibitor of the Wnt pathway, acting by activating the destruction complex (Venerando et al., 2013) and, thus, leading to the destabilization of β‐catenin through its phosphorylation and subsequent degradation.

Consistently, we observed that pyrvinium pamoate determined a decrease in the β‐catenin total protein levels in a time‐ and dose‐dependent manner (Figures 3a and 3b, respectively).

Figure 3.

Figure 3

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Time‐ and dose‐dependent modulation of β‐catenin expression under pyrvinium pamoate treatment. (a) MSTO‐211H cells were treated with pyrvinium pamoate at its IC50 value for 24, 48, and 72 hr and β‐catenin levels were assessed by Western blot analysis. Control cells (CTR) were treated with DMSO alone for the indicated time points. An anti‐GAPDH antibody was used for a loading control. (b) MSTO‐211H cells were treated for 72 hr with increasing doses of pyrvinium pamoate. Control cells were treated with DMSO alone. The cell lysates were then analyzed for β‐catenin expression by Western blotting. An anti‐GAPDH antibody was used for a loading control

3.3. Pyrvinium pamoate decreases the expression of downstream genes in the Wnt signaling pathway in mesothelioma cells

To further confirm the inhibition of the Wnt pathway in MM cells upon pyrvinium pamoate treatment, we examined the effects of this drug on the expression of well‐established Wnt target genes in MSTO‐211H cells by real‐time qRT‐PCR. In particular, we analyzed pyrvinium pamoate effects on genes both in the β‐catenin‐dependent canonical Wnt pathway (AXIN2 and CCND1, encoding cyclin D1) (Jho et al., 2002; Katoh & Katoh, 2007) and in the β‐catenin‐independent non‐canonical Wnt pathway (receptor tyrosine kinase like orphan receptor 2, ROR2) (Ford, Qian Ma, Quadir, & Ward, 2013). Consistent with the inhibition of this pathway, the transcripts of AXIN2, CCND1 and ROR2 genes were down‐regulated after 24 hr of treatment with pyrvinium pamoate in a dose‐responsive manner (Figure 4a), although ROR2 down‐regulation did not reach statistical significance. We also analyzed the expression of these Wnt target genes at 48 and 72 hr after treatment with pyrvinium pamoate at its IC50 value and found a down‐regulation of all the analyzed genes in a time‐dependent manner (Figure 4b).

Figure 4.

Figure 4

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Pyrvinium pamoate decreased the expression of downstream genes in the Wnt pathway in a dose‐ and time‐dependent manner. (a) Increasing concentrations of pyrvinium pamoate were used for 24 hr in MSTO‐211H cells. Transcripts of AXIN2, CCND1, and ROR2 genes were then analyzed by real‐time qRT‐PCR. Gene expression in treated cells were calculated by the 2−ΔΔCt method relatively to control cells (CTR) treated with DMSO alone. Data are reported as means ± standard deviations of three independent experiments. Statistical analysis was performed by subjecting the ΔCt values of treated and control samples to one‐way Anova with Dunnett post‐test, to compare all data vs control. Statistically significant differences are indicated with: *significant (p < 0.05). (b) MSTO‐211H cells were treated for 48 and 72 hr with pyrvinium pamoate at its IC50 value and then the expression of the above mentioned genes was analyzed by real‐time qRT‐PCR. Gene expression in treated cells were calculated by the 2−ΔΔCt method relatively to control cells treated with DMSO alone. Data are reported as means ± standard deviations of three independent experiments. Statistical analysis was performed by subjecting the ΔCt values of treated and control samples to Student t‐test. Statistically significant differences are indicated with: *significant (p < 0.05) and **very significant (p < 0.01)

3.4. Pyrvinium pamoate decreases the expression of key genes in mesothelioma biology

Once we confirmed that pyrvinium pamoate targets the Wnt/β‐catenin pathway also in MM, we aimed to analyze the modulation of some important MM‐related genes, whose expression can be regulated by the Wnt/β‐catenin pathway but that were never investigated as targets of pyrvinium pamoate. In details, we analyzed, through real‐time qRT‐PCR, the expression of the following genes: VEGFA (vascular endothelial growth factor A), encoding a potent mitogen for the vascular endothelium, also known to be an autocrine growth factor for MM (Strizzi et al., 2001); MET, encoding a tyrosine kinase receptor contributing to MM aggressiveness, by activating intracellular survival pathways (Garajová, Giovannetti, Biasco, & Peters, 2015); HMGB1 (high mobility group box 1) and its downstream target MMP9 (matrix metallopeptidase 9), which have key roles in survival and invasion of MM cells (Carbone et al., 2012). We observed that treatment with pyrvinium pamoate at its IC50 value for 72 hr reduced the expression of all these genes in MSTO‐211H cells (Figure 5).

Figure 5.

Figure 5

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Pyrvinium pamoate decreased the transcripts of key genes in mesothelioma biology. Changes in expression levels of VEGFA, MET, HMGB1, and MMP9 genes were assessed in MSTO‐211H cells by real‐time qRT‐PCR analysis after 72 hr of treatment with pyrvinium pamoate at its IC50 value. Gene expression in treated cells were calculated by the 2−ΔΔCt method relatively to control cells (CTR) treated with DMSO alone. Data are reported as means ± standard deviations of at least four independent experiments. Statistical analysis was performed by subjecting the ΔCt values of treated and control samples to Student's t‐test. Statistically significant differences are indicated with: *significant (p < 0.05), **very significant (p < 0.01) and *** extremely significant (p < 0.001)

3.5. Pyrvinium pamoate reduces the migration ability of mesothelioma cells

We subsequently examined the effect of pyrvinium pamoate on cell migration ability through a scratch test on MSTO‐211H cells. In particular, we made a scratch in the cell monolayers and evaluated cell ability to close the wound upon 24 and 48 hr of treatment with pyrvinium pamoate at its 72‐hr IC50 value and at 1/2 of this value. We found that both drug concentrations affected cell migration of treated cells, impairing their ability to heal the wound compared with control cells (Figure 6). We observed that the number of viable cells was not significantly affected by pyrvinium pamoate at the culture times used for the scratch assay, through trypan blue staining of MSTO‐211H cells identically treated in parallel experiments (data not shown).

Figure 6.

Figure 6

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Pyrvinium pamoate impaired mesothelioma cell migration ability. A scratch was created onto confluent monolayers of MSTO‐211H cells. These cells were allowed to migrate into the wound over the subsequent 24 and 48 hr of treatment with pyrvinium pamoate, at the indicated concentrations, or with DMSO, as a control (CTR). Migration was significantly inhibited by pyrvinium pamoate treatment. The showed micrographs are representative of three independent experiments

3.6. Pyrvinium pamoate inhibits mesothelioma spheroid formation

We also investigated the tumor suppressive activity of pyrvinium pamoate by evaluating the potential ability of this drug to suppress the formation of multicellular tumor spheroids (MCTS), which are three‐dimensional cultures of cancer cells that simulate more accurately the in vivo behavior of cells in tumor tissues compared to two‐dimensional cell cultures and represent an in vitro model of the first step of metastasis formation (Friedrich, Ebner, & Kunz‐Schughart, 2007; Soriţău et al., 2010). To this purpose, we cultured MSTO‐211H cells under nonadherent conditions in presence of a sub‐toxic concentration of pyrvinium pamoate. We observed that the drug abolished the ability of MSTO‐211H cells to form spheroids (Figure 7).

Figure 7.

Figure 7

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Pyrvinium pamoate inhibited the formation of spheroids. MSTO‐211H cells are able to form spheroids in low‐attachment conditions within 3 days from seeding. The treatment with pyrvinium at a sub‐toxic concentration completely inhibited tumor spheroid formation (original magnification 40×)

4. DISCUSSION

MM is a highly treatment‐resistant malignancy, mainly associated with exposure to asbestos (Rom & Palmer, 1974; Thomson, 1963; van Meerbeeck & Damhuis, 2011). Despite the numerous findings in the pre‐clinical research, little advances have been achieved in the clinical practice and, therefore, new pharmacologic approaches are urgently needed.

Drug repositioning, using already approved drugs for new indications, is a promising strategy to identify active molecules for a more rapid and less expensive clinical translation (Abruzzese et al., 2017). Among these molecules, the anti‐helminthic drug, pyrvinium pamoate, is emerging as a safe compound active against cancer models in vitro and in vivo (Esumi, Lu, Kurashima, & Hanaoka, 2004; Wiegering et al., 2014; Xiang et al., 2015; Xiao et al., 2016; Xu et al., 2013; Zhang et al., 2017; Zheng, Liu, & Pan, 2017). Indeed, at doses of 35 mg/kg, pyrvinium pamoate is safe and without systemic side effects (Smith, Kinkel, Gryczko, & Goulet, 1976). Considering the previously established inhibitory effect of pyrvinium pamoate on the Wnt/β‐catenin pathway and given the role of this pathway in MM development and resistance to therapies (de Assis & Isoldi, 2014; Fox et al., 2013; He et al., 2005; Perumal et al., 2017), in this study we explored, for the first time, the effects of pyrvinium pamoate on MM cells. We observed that this compound efficiently inhibited the proliferation of a panel of MM cell lines and reduced MM cell ability both to migrate and to grow as multicellular spheroids under nonadherent conditions.

At the molecular level, pyrvinium pamoate reduced β‐catenin expression in a time‐ and dose‐dependent manner and down‐regulated downstream genes in the Wnt/β‐catenin pathway in MM cells. In particular, we observed a decreased expression of CCND1 gene, which encodes cyclin D1, an important regulator of cell cycle progression. Excessive cyclin D‐cyclin dependent kinase (CDK) expression and/or activity, which leads to dysregulated cell growth, is a common event in different cancer types, including MM (de Assis, Locatelli, & Isoldi, 2014; Kettunen et al., 2001; Qie & Diehl, 2016). Thus, the observed CCND1 gene down‐regulation in MM cells upon treatment with pyrvinium pamoate is consistent with its anti‐proliferative effect on these cells. Moreover, pyrvinium pamoate decreased the expression of another gene in the Wnt/β‐catenin pathway, AXIN2. Although this gene is a negative regulator of this pathway, the observed down‐regulation of AXIN2 following pyrvinium pamoate treatment is, however, consistent with the inhibition of the Wnt pathway. Indeed, AXIN2 participates in a negative feedback loop whereby the Wnt signaling induces AXIN2 expression to limit the duration or intensity of a Wnt‐initiated signal (Jho et al., 2002). Therefore, the observed AXIN2 down‐regulation is indicative of Wnt pathway inhibition. We also analyzed the expression of ROR2, an important mediator of the β‐catenin‐independent non‐canonical Wnt pathway (Katoh & Katoh, 2007, 2017), and observed its down‐regulation upon pyrvinium pamoate treatment. ROR2 is recently emerging as a new target for anti‐cancer therapeutic intervention, owing to its expression in several cancers, in which it strongly correlates with more aggressive disease states (Debebe & Rathmell, 2015; Yang et al., 2017). Moreover, in some cancer models, ROR2 has been demonstrated to be involved in migration, invasion, (Arabzadeh, Hossein, Salehi‐Dulabi, & Zarnani, 2016; Henry, Llamosas, Djordjevic, Hacker, & Ford, 2016) and metastasis (Lai et al., 2012). Nevertheless, the expression and function of ROR2 have never been investigated in MM and deserve further investigation.

We then analyzed the expression of some Wnt target genes of particular interest for MM biology. Among the numerous genes potentially targeted by the Wnt inhibitor pyrvinium pamoate, we first focused on HMGB gene. This gene has recently emerged as an important determinant in the initiation and progression of MM (Carbone et al., 2012) and it has been demonstrated to be expressed in response to Wnt signaling activation (Reed et al., 2016). In MPM cells, inhibition of HMGB1 determines a decrease in motility, survival and anchorage‐independent growth (Jube et al., 2012). For these reasons, we analyzed the expression of HMGB1 and its target gene MMP9, whose product is known to degrade almost all the extracellular matrix components and to play a crucial role in tumor invasion and metastasis. Interestingly, our results indicate that pyrvinium pamoate can down‐regulate both HMGB1 and MMP9 expression in MM cells. The decrease in the expression of these genes upon pyrvinium pamoate treatment seems consistent with the observed cellular effects of this drug on both motility and spheroid formation under nonadherent conditions (Lee et al., 2010).

MET gene transcribes for a tyrosine kinase receptor that, when activated by its ligand, the Hepatocyte Growth Factor (HGF), promotes cell growth, motility and angiogenesis. MET has been identified as a target of the Wnt signaling cascade (Boon, van der Neut, van de Wetering, Clevers, & Pals, 2002) and has been demonstrated to be expressed in the majority of MPMs (Bois et al., 2016; Jagadeeswaran et al., 2006; Tolnay et al., 1998). High HGF levels were found in over 90% of all the pleural effusions from patients affected by tumors within the pleura and adjacent lung tissue (Eagles et al., 1996) and in 60% of MPMs (Jagadeeswaran et al., 2006). The HGF/c‐MET signaling pathway strongly contributes to MM aggressiveness, by activating its downstream intracellular effectors, such as NF‐kB, PI3K/AKT, STAT, MAPK (Garajová et al., 2015). For this reason, its inhibition has been recognized as a potential therapeutic strategy for MM treatment. In our study we found a down‐regulation of this important player of MM aggressiveness upon pyrvinium pamoate treatment of MM cells.

Finally, we analyzed the expression of VEGFA, encoding a potent mitogen for the vascular endothelium, also known to be an autocrine growth factor for MPM (Strizzi et al., 2001). Overexpression of VEGFA has been related to poor prognosis of MPM (Hirayama et al., 2011; Yasumitsu et al., 2010). Importantly, our experiments show that pyrvinium pamoate is able to down‐regulate VEGFA expression in MM cells.

In conclusion, our data show that pyrvinium pamoate affects hallmark features of MM cells, impairing both their growth and migration. Our findings suggest that the inhibition of the Wnt/β‐catenin pathway could, at least in part, underlie pyrvinium pamoate cellular effects. However, further studies are needed to evaluate other possible mechanisms of action of the drug, especially in MM cells in which the Wnt/β‐catenin pathway is inactive, such as NCI‐H28 cells, carrying a β‐catenin deletion (Shigemitsu et al., 2001). Indeed, considering the previously established role of pyrvinium pamoate in inhibiting the AKT pathway (Carrella et al., 2016; Venerando et al., 2013) and given the crucial role of the AKT kinase in MM cell survival (Indovina et al., 2012; Pentimalli et al., 2018), the inhibition of the AKT pathway could contribute to the pyrvinium pamoate effects in MM cells. Overall, although our findings should be confirmed in other MM cell lines and the molecular mechanisms underlying pyrvinium pamoate effects on MM cells require further investigation, our study suggests that the repurposing of pyrvinium pamoate for MM treatment could represent a promising approach.

CONFLICTS OF INTEREST

The authors declare that there are no conflicts of interest.

ACKNOWLEDGMENTS

This work is dedicated to the memory of Mr. Vittorio Stortini. The authors are thankful to Lassi family for their contribution. This work was supported by Commonwealth of Pennsylvania, Associazione Italiana per la Ricerca sul Cancro (grant no. IG2014‐15690), Sbarro Health Research Organization (http://www.shro.org), and Mesothelioma Applied Research Foundation (grant no. 2016).

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