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Synergistic Activity of the c-Met and Tubulin Inhibitor Tivantinib (ARQ197) with Pemetrexed in Mesothelioma Cells

Leticia G. Leon1,2,*

, Maria Gemelli3, Rocco Sciarrillo

2,4, Amir Avan

2,5, Niccola Funel

6 and

Elisa Giovannetti2,6

1Instituto de Tecnologias Biomedicas, Center for Biomedical Research of the Canary Islands, University of La Laguna,

Spain; 2Dept. Medical Oncology, VU University Medical Center, Amsterdam, The Netherlands;

3Faculty of Medicine,

University of Milano, Milano, Italy; 4Dept. Haematology, VU University Medical Center, Amsterdam, The Netherlands;

5Molecular Medicine group, Department of Modern Sciences and Technology, Faculty of Medicine, Mashhad University

of Medical Science, Mashhad, Iran; 6Start-Up Unit, University of Pisa, Pisa, Italy

Abstract: Malignant pleural mesothelioma (MPM) is a lethal disease with scarce therapeutic options, and preclinical stud-

ies on new targeted-agents are warranted. Because previous studies reported high c-Met expression and alterations in the

microtubules network in most MPM samples, we evaluated the activity of tivantinib, which has been recently suggested to

affect microtubule polymerization in addition to inhibiting c-Met. In four MPM cell lines tivantinib inhibited both c-Met

activity and microtubule polymerization, resulting in inhibition of cell-growth with IC50s ranging between 0.3 M

(MSTO-211H) and 2.4 M (H2052). Furthermore tivantinib synergistically enhanced the antiproliferative and pro-

apoptotic activity of pemetrexed, as detected by sulforhodamine-B-assay and flow cytometry. The synergistic interaction

was associated with reduction of thymidylate synthase expression and inhibition of migratory activity. In aggregate, these

data show the ability of tivantinib to specifically target key pathways in MPM cells and synergistically interact with pe-

metrexed, supporting further studies on this therapeutic approach.

Keywords: c-Met, malignant pleural mesothelioma, migration, pemetrexed, synergistic interaction, tivantinib, tubulin.

INTRODUCTION

“Malignant pleural mesothelioma (MPM) is an aggres-sive, lethal cancer of the pleura, associated with exposure to asbestos, and is increasing in incidence in Europe”, as re-ported previously [1, 2]. Only a minority of patients are eli-gible for radical surgery. The majority (85-90%) of MPM patients present with stage III/IV tumors, as evidenced by a series staged by positron emission tomography (PET), that identified 3% stage I, 9% stage II, 48% stage III and 40% stage IV [3]. These patients rely on palliative treatments, aiming at survival prolongation and palliation.

Most chemotherapeutic agents exhibit low intrinsic activ-ity; an exception is pemetrexed [4]. The combination of pe-metrexed and cisplatin has become the standard first-line therapy after a phase III trial showing improved response rate (RR, 41.3%), time-to-progression (TTP, 5.7 months), overall survival (OS, 12.1 months), and quality of life, when compared with cisplatin monotherapy [5]. Pemetrexed-carboplatin is also a well-tolerated and active regimen. In-deed, in a phase II trial, “102 MPM patients showed a dis-ease control rate (65%), TTP (6.5 months), and OS (12.7 months) similar to the results achieved with the standard regimen of pemetrexed-cisplatin", as reported previously [6]. Nonetheless the overall outcome of this disease remains poor

*Address correspondence to this author at the Center for Biomedical Re-

search of the Canary Islands, Instituto de Tecnologias Biomédicas, Univer-

sity of La Laguna, Campus de la Salud s/n, 38071 La Laguna, Tenerife,

Spain; Tel: +34-619538827; Fax: 922319279;

E-mail: lgleon@ull.edu.es

and most patients experience tumor progression or relapses within a year. A number of prognostic factors have been described [7, 8], but discovery of new effective targets and drugs is urgently warranted.

Given the heterogeneous and complex genetic nature of MPM [9, 10], many molecular aberrations underlying its progression and affecting clinical response have to be identi-fied, while several trials of new biological agents in combi-nation with pemetrexed and platinum are currently ongoing (http://www.clinicaltrials.gov). Previous studies supported preclinical investigations on key kinases that act as central regulators of the neoplastic process and are amenable to pharmacological inhibition. However, treatment of MPM patients with the epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors (TKIs) failed to improve response and survival [4, 11]. Similarly, phase II trials of the anti-angiogenic TKIs SU5416, vatalanib, and sorafenib demon-strated only modest activity [12]. The lack of the expected positive outcome may have been caused by the lack of pa-tient selection, which should be based on pivotal predictive markers, or by the lack of the right chemotherapy corner-stone for the addition of these targeted therapies.

Several studies suggested that the c-Met receptor tyrosine kinase is a promising candidate biomarker and target in MPM [13-18]. The expression of c-Met protein has been detected by immunohistochemistry in 70% to 100% of paraf-fin-embedded MPM samples but not in normal mesothelial cells [13, 14], and c-MET gene mutations have been reported in about 16% of MPM patients [15]. Moreover, c-Met

1330 Current Drug Targets, 2014, Vol. 15, No. 14 Leon et al.

plasma membrane localization recently emerged as a rele-vant prognosis biomarker in MPM [19]. This should prompt further studies on the role of c-Met expression, activation state, and subcellular localization on the efficacy of both chemotherapy and c-Met targeted therapies.

The c-Met inhibitors, including both small molecules TKIs and monoclonal antibodies, represent an important new class of drugs in the battle against advanced non-small-cell lung cancer [20]. However, MET has been implicated in a variety of human malignancies, both as a necessary onco-gene and as a subsidiary gene in the malignant metastatic behaviour, offering a compelling rationale for strategies tar-geting c-Met in many other tumors, such as for example gas-tric and pancreatic cancers [21-23].

In particular, considerable efforts have been made in the development of effective TKIs of c-Met, and a large number of new molecules such as tivantinib (ARQ197), crizotinib (PF-02341066), cabozantinib (XL184, BMS-907351), golvatinib (E7050), MGCD265, JNJ-38877605, MK-2461, EMD 1204831, EMD 1214063, INCB028060 and AMG 208, are in preclinical and clinical on-going investigations [20, 21]. Among these compounds, tivantinib, cabozantinib and crizo-tinib are part of more advanced clinical development.

Of note, two recent studies suggested that tivantinib in-hibits microtubule polymerization in addition to inhibiting c-Met, suppressing the growth of both c-MET-addicted and not addicted cancers [24, 25]. These findings are extremely in-teresting in the context of MPM because microarray data from MPM samples showed the cytoskeleton/spindle micro-tubules network was the second-most significantly affected network [26]. This study demonstrated also that a non-taxane small molecule inhibitor, epothilone B, was able to decrease the viability of fourteen MPM cell lines, suggesting the po-tential of non-taxane microtubule-targeting therapy for the treatment of MPM.

Therefore, the aim of the current preclinical study was to evaluate the activity of tivantinib as well as the molecular and cellular characteristics underlying the interaction be-tween pemetrexed and tivantinib in MPM cells. We found a potent synergistic interaction between pemetrexed and ti-vantinib against four human MPM cell lines. Several factors including apoptosis induction, modulation of phosphoryla-tion and expression of critical gene products involved in drug activity, but also perturbation of microtubule dynamics, con-tributed to this synergistic interaction. Moreover we ob-served a significant reduction in cell migration, further sup-porting the ongoing prospective, open- phase Ib trial of ti-vantinib in combination with pemetrexed and carboplatin as first-line therapy in patients with advanced MPM [Clinical-Trials.gov Identifier: NCT02049060].

METHODS

Cells & Chemicals

The human MPM cell lines, NCI-H2452 (epithelioid), NCI-H28 and NCI-H2052 (sarcomatoid), and MSTO-211H (biphasic), were purchased from ATCC (Manassas, VA, USA).

All these cells were cultured in RPMI-1640 medium, containing, supplemented with 2 mM L-glutamine, 10%

heat-inactivated fetal bovine serum, and 1% penicillin–streptomycin (10,000 U/mL), at 37ºC, in 5% CO2. The cells were cultured in 25 and 75 cm

2 culture flasks (Greiner-Bio-

One, Frickenhausen, Germany) and harvested with trypsin (Invitrogen, Paisley, UK), as described previously [23]. Ti-vantinib was purchased from Bio-Connect (Huissen, The Netherlands), while pemetrexed was a kind gift from Eli Lilly and Company (Indianapolis, IN), as described in our previous studies [22, 27].

Analysis of c-MET Copy Number and Gene Expression of Determinants of Drug Activity

Previous studies showed no genetic alterations within the

semaphorin domain (N375S, M431V, and N454I), the jux-tamembrane domain (T1010I and G1085X), as well as no

alternative splice product skipping of entire exon 10 for the

c-MET gene in the NCI-H28, NCI-H2052, NCI-H2452 and MSTO-211H cells [15]. In the present study we evaluated

the copy number of the c-MET gene with a specific

TaqMan® copy-number assay (Hs04933308_cn, Life Tech-nologies, Applied Biosystems, Foster City, CA, USA), using

the ribonuclease P RNA component H1 (RPPH1) gene as

endogenous control. The primers of the c-MET gene were labeled with FAM probe; the RPPH1 gene was labeled with

VIC probes. PCR was operated on the ABIPRISM-7500

instrument. The copy number of c-MET was analyzed by the CopyCaller v1.0 software (Applied Biosystems) and cali-

brated to the copy number of this gene in the Reference Ge-

nomic DNA (Promega, Mandison, WI, USA), which is as-sumed to carry two copies of the c-MET gene. Furthermore,

we used as a positive control the genomic DNA of the

HCC827-GR5 lung cancer cells (kindly provided by Dr. Pasi Janne, Dana-Farber Institute, Boston, MA), carrying a ampli-

fication from 7q31.1 to 7q33.3, which encompasses c-MET

but not the gene encoding HGF. PCRs were performed on the ABIPRISM-7500 instrument, as described previously

[22].

Gene expression of the following pivotal determinants of

drug sensitivity/resistance was assessed by reverse tran-

scriptase quantitative polymerase chain reaction (RT-QPCR): c-MET (NM_000245.2), thymidylate synthase (TS,

(NM_0010711), Folylpolyglutamate-sintase (FPGS,

NM_004957), and reduced folate carrier (RFC, NM_194255.1). Moreover, we studied whether single drugs

and their combinations, at IC50s, modulated expression of the

primary target of pemetrexed, TS. RNA was extracted using the QiaAmp RNA mini-Kit (Qiagen), and reverse transcribed

to cDNA. Primers and probes were obtained from Applied

Biosystems [27, 28], and PCR data were normalized to the housekeeping gene -actin. Quantification of gene expres-

sion was obtained using reference standard curves from dilu-

tions of Quantitative-PCR Human-Reference Total-RNA (Stratagene, La Jolla, CA, USA). The data from these basal

expression studies were related to chemosensitivity, while

measurement of gene expression in treated versus untreated cells was performed using the CT calculation, where CT

is the threshold cycle. The expression of the target genes,

normalized to -actin, and relative to untreated cells (calibra-tor), was calculated as 2

- CT and reported as percentage of

variation versus untreated cells, as described previously [28].

Tivantinib & Pemetrexed in MPM Current Drug Targets, 2014, Vol. 15, No. 14 1331

Immunoblotting and Enzyme-Linked Immunosorbent Assay (ELISA)

To evaluate the modulation of c-Met and phospho-c-Met expression, Western blot analyses were performed in cells treated for 2 hours with 1 μmol/L tivantinib, after short stimulation (i.e. 10-minute exposure) to 40 ng/mL of the c-Met ligand hepatocyte growth factor (HGF, Sigma Chemical Co., Zwijndrecht, The Netherlands), as described previously [29]. Membranes were incubated with the anti-c-Met (c-12, 1:1,00 dilution; Santa Cruz Biotechnology,) and anti-phospho-Y1003-c-Met (i.e., Cbl-binding site, 1:1,00 dilu-tion; BioSource International Inc., Camarillo, CA, USA), and mouse anti- -actin (1:50,000; Sigma). Membranes were probed for 1 hour with goat anti-rabbit-InfraRedDye® 800 Green (1:10000; #926-32211; Westburg, Leusden, The Netherlands) and goat anti-mouse-InfraRedDye® 680 Red (1:10000; #926-32220), as described previously [23]. Fluo-rescent proteins were detected by an Odyssey Infrared Imager (LI-COR Biosciences Lincoln, Nebraska USA), at 84 μm resolution, using high quality settings), and semi-quantitative analysis of the bands was performed with the Odyssey version 3.0 software, as described previously [23].

The analysis of tivantinb and pemetrexed effects on c-Met activity was further studied by specific ELISA phos-phorylation assays. MPM cells were treated for 2 hours with IC50s of single drugs and their combinations after short stimulation with HGF (40 ng/ml), as described [29]. After protein extraction from cell pellets, c-Met phosphorylation at tyrosine residues 1230, 1234, and 1235 (i.e., c-Met auto-phosphorylation sites [pYpYpY1230/34/35]) was evaluated with specific ELISA assays (Life Techonologies, As-say#KHO0281), and normalized respectively to the total c-Met, as well as to protein content [28].

Cell Growth Inhibition Assays

Cell growth inhibitory effects of the drugs (after 72-hour exposure to 0.001–50 μM concentrations) were studied using the sulforhodamine-B (SRB) assay. For this assay, cells were plated at 5x10

4 cells per well and the inhibition of cell

growth was expressed as the percentage of control (untreated cells) absorbance (corrected for absorbance before drug ex-posure). After 72 hour-exposure, cells were processed for the SRB assay, as described previously [28]. The 50% inhibitory concentration of cell growth (IC50) was calculated by non-linear least squares curve fitting (GraphPad PRISM version 5.0, Intuitive Software for Science, San Diego, CA, USA).

Analysis of the Pharmacological Interaction of Tivantinib

with Pemetrexed

To analyze the pharmacological interaction of tivantinib and gemcitabine, the cell growth inhibition of each single drug was compared with the combination, using the combi-nation index (CI), where CI values of <0.9, 0.9-1.1, and >1.1 indicated synergistic, additive and antagonistic interactions, respectively [23]. All the combination studies were per-formed with simultaneous treatments, testing six increasing concentrations of pemetrexed, and a fixed concentration of tivantinib (10 μM). The median-drug effect analysis method was used for analyzing the drug interaction, as described previously [30].

Growth inhibition values lower than 50% were not con-sidered as relevant, while CIs at fraction affected (FA) of 0.5, 0.75 and 0.9 were measured and averaged for each ex-periment. These CIs s were then used to obtain the average between triplicate experiments. The software CalcuSyn (ver-sion 2.0) was used for the analysis of the data (Biosoft, Ox-ford, UK).

Analysis of Cell Cycle and Cell Death

Cells were seeded in a six-well plate at a density of 2 105

cells/well. After 24 hours the compounds at IC50s were added and the cells were incubated for 72 hours. Cells were then harvested, transferred to 12 75 mm tubes (Becton Dickinson, San José, CA, USA) and collected by centrifuga-tion at 1,000 rpm for 10 minutes at 5ºC. The supernatant was discarded while the pellets were resuspended in 500 μL of propidium iodide buffer (PI 50 g/mL, in 0.1% sodium cit-rate, 0.1 mg/mL RNAse and 0.1% triton X-100). Flow cy-tometry analysisof DNA content (using 10,000 cells/sample) was performed using FACSCalibur (Becton Dickinson), equipped with theFACSdiva version 6.0 software (Becton Dickinson) and FlowJo version 7 software (Treestar, Ash-land, OR, USA).

Microtubule Stabilization Analysis

Cells were seeded in a six well-plate (1 105 cells/well)

and exposed to drugs for 24 hours. Then cells were harvested and centrifuged at 400 g for 5 minutes. The supernatant was removed by aspiration, and the cells were fixed in 1 mL of 0.5% glutaraldehyde in MicroTubule Stabilizing Buffer (MTSB:80 mM Pipes [pH 6.8], 1 mM MgCl2, 5 mM EDTA, and 0.5% Triton X- 100 [TX-100]). After a 10 minutes incu-bation at room temperature, glutaraldehyde was quenched by the addition of 0.7 ml of 1 mg /ml NaBH4 in phosphate buffered saline (PBS). The cells pellets were obtained with centrifugation at 600 g, for 5 minutesand then resuspended in 20 ml of 50 mg /ml RNase an in Antibody Diluting Solution (AbDil: PBS [pH 7.4], 0.2% TX-100, 2% bovine serum al-bumin [BSA], and 0.1% NaN3) and incubated for 12 hours at 4 ºC. Then, 5 mL of a 1:50 dilution of anti-tubulin anti-body in AbDil was added (achieving a final dilution of 1:250), and the cells were incubated for additional 3 hours in the dark.

After centrifugation at 300 g for 5 minutes, the cell pel-lets were resuspended in 0.2 mL of AbDil, and 2.5 mL of fluorocrome AlexaFluor® 488 (Invitrogen, Life Technolo-gies) was added [31]. Then cells were incubated for other 45 minutes. Finally, the cells were also stained with 0.5 mL PI buffer and incubated in the dark on ice during 30 minutes. Samples were analyzed by flow cytometry, and tubulin lev-els were calculated considering the average of the antibody– FITC fluorescence, normalized to a value of 100 and com-pared to the untreated controls.

Wound Healing Assay

To assess the migration into the wound area after drug exposure, the MPM cells were plated (with a density of 8 10

4 cells/well) in 96 wells plates. After 24 hours, we cre-

ated artificial wound tracks in the confluent monolayers us-ing a specific scratcher, as described previously [23]. The

1332 Current Drug Targets, 2014, Vol. 15, No. 14 Leon et al.

cells were washed with PBS for removal of the detached cells and exposed to 10 μM tivantinib, pemetrexed at IC50, or their combination. Images were taken at the start of the exposure (so-called time 0), and then after 4, 8, 12, 16, 20 and 24 hours. Migration was analyzed using the Lei-caDMI300B (Leica Microsystems, Wetzlar, Germany) mi-gration station equipped with the Scratch Assay version 6.1 software (Digital Cell Imaging Labs, Keerbergen, Belgium), as described previously [32].

Statistical Analysis

All the experiments were performed in triplicate and were repeated at least twice. Statistical analyses were carried out using GraphPad Prism (Intuitive Software for Science). Results are expressed as mean values ± standard deviation (SD) or standard error of the mean (SEM) for the indicated number of independent measurements.

Differences between the average values recorded for dif-ferent experimental conditions were evaluated by Student’s

t-test or ANOVA followed by the Tukey's multiple compari-son test. The level of significance was set at P<0.05 and P values are reported in the figures and in their legends. RESULTS

Genetic Background of the Human MPM Cell Lines

Genomic DNA and RNA extracted from the various MPM cells were used to evaluate MET copy number and mRNA expression levels of genes potentially affecting drug activity, respectively. As reported in the Fig. (1A-B), all the MPM cell lines have the same copy number, while the mRNA expression of c-Met is slightly higher in the H2052 and MSTO-211H compared to H2452 and H28 cells. Simi-larly, the highest values of TS expression were detected in the H2052 and MSTO-211H cells. In the H2052 and MSTO-211H cells we observed the two strongest bands for phos-pho-Y1003-c-Met, as depicted in the blots in the Fig. (1C), while the basal levels of c-Met phosphorylation at tyrosine residues 1230, 1234, and 1235 were slightly higher in H28 and MSTO-211H cells (Fig. 1D).

Fig. (1). Genetic background of the human MPM cell lines. (A) c-MET gene copy number determined by quantitative PCR in MPM cell

lines. The c-MET–amplified HCC827GR5 (c-MET copy number=10N) cells were used as positive control. (B) TS, FPGS, RFC and c-Met

mRNA expression as determined by qRT-PCR in MPM cell lines. Quantification of gene expression was performed using standard curves

obtained with dilutions of cDNA from Quantitative-PCR Human-Reference Total-RNA (Stratagene), and preliminary validation experiments

were performed to demonstrate that the efficiencies of the targets and reference ( -actin) gene amplifications were approximately equal. Col-

umns, means from three independent experiments; bars, SEM. (C) Representative Western blot of c-Met expression in the MPM cell lines. A

145-KDa protein band corresponding to the proteolitically processed (biologically active) form of c-Met, phosphorylated at Y1003 was

clearly detected in whole-cell protein extracts from MSTO-211H and H2052 cells, while much fainter bands were observed in H2425 and

H28 cells. Per lane 30 g of protein was loaded. As a loading control, -actin levels are indicated. (D) Phosphorylation of c-Met normalized

to total c-Met and protein content, assessed by ELISA assays with standard curves which were run with each specific assay using 100, 50,

25, 12.5, 6.25, 3.12, and 1.6 units/mL of standard human c-Met [pYpYpY1230/1234/1235], and 50, 25, 12.5, 6.25, 3.12, and 1.6 ng/mL of

standard recombinant human c-Met. Values calculated from these standard curves were then normalized for total protein content. Columns,

means from two independent experiments; bars, SEM.

Tivantinib & Pemetrexed in MPM Current Drug Targets, 2014, Vol. 15, No. 14 1333

Because of the different histological background, and high c-Met expression H2052 and MSTO-211H cells were selected for most of the following experiments to gain fur-ther insight into potential molecular mechanisms regulating drug interactions between tivantinib and pemetrexed.

Modulation of TS Expression and Phospho-c-Met

Remarkably, the PCR data showed that H2052 and MSTO-211H cells treated with tivantinib had a 5-fold de-crease in TS mRNA expression (Fig. 2A). Conversely, pe-metrexed caused a significant increase of TS levels in the MPM cells. Moreover, the combination of pemetrexed and tivatinib was able not only to prevent the increase of TS cause by pemetrexed, but also to decrease the mRNA ex-pression of TS. Similarly, both tivantinib and its combination with pemetrexed significantly reduced the phosphorylation status of c-Met (Fig. 2B), supporting also in these MPM cells the anti-c-Met activity of tivantinib through disruption of the ligand-mediated (HGF) c-Met phosphorylation, as described in other cancer cell lines [33].

Fig. (2). Modulation of TS expression and phospho-c-Met. (A)

Modulation of TS mRNA expression by tivantinib, pemetrexed and

their combination in MSTO-211H and H2052 cells, as detected by

qRT-PCR. Columns, mean values obtained from three independent

experiments; bars, SD. *P<0.05, significantly different from con-

trols. (B) Modulation of c-Met phosphorylation by tivantinib, pe-

metrexed and their combination in MSTO-211H and H2052 cells,

as detected by specific ELISA assays. Columns, mean values ob-

tained from two independent experiments; bars, SEM. *P<0.05,

significantly different from controls.

Antiproliferative and Synergistic Activity of Tivantinib

and Pemetrexed

A dose-dependence inhibition of tumor cell growth was observed with tivantinib and pemetrexed in all the four MPM

cell lines. The IC50s values of tivantinib ranged from 0.31±0.04 μM to 2.4 μM in MSTO-211H (Fig. 3A) and H2052 cells, respectively. In H28 and H2452 cells IC50s val-ues of tivantinib were 2.0±0.04 and 1.2±0.02 μM.

MSTO-211H cells were also the cells most sensitive to pemetrexed (Fig. 3A), whereas H28 cells were the most in-herently drug resistant to both pemetrexed and carboplatin, as described previously [28]. Using a ratio of concentrations at which drugs are equipotent, the simultaneous combination of tivantinib and pemetrexed reduced the IC50 values of both drugs in MSTO-211H cells, as illustrated by the representa-tive growth inhibition curve in Fig. (3A).

However, since tivantinib is administered orally and reaches relatively stable plasma concentration for a pro-longed time, with concentration ranging between 2.7 and 27 μM after twice per day administration of 100-400 mg [34], we performed pharmacological interaction studies using ti-vantinib at a fixed concentration of 10 μM. At this variable ratio the synergism was most pronounced in H2052 than in MSTO-211H cells, with average CI values of 0.37 and 0.70, respectively (Fig. 3B).

Fig. (3). Cytotoxicity and pharmacological interaction of ti-

vantinib, pemetrexed and their combination. (A) Representative

curves of growth inhibitory effects of tivantinib, pemetrexed and

their combination (at fixed IC50 ratio) in MSTO-211H cells (for the

combinations drug concentrations on the X-axis are referred to

pemetrexed). Points, mean values obtained from three independent

experiments; bars, SEM. (B) Mean CI values of all the combina-

tions in MSTO-211H and H2052 cells, using a fixed concentration

of tivantinib (10 μM). Columns, mean values obtained from three

independent experiments; bars, SEM.

1334 Current Drug Targets, 2014, Vol. 15, No. 14 Leon et al.

Cell Cycle Perturbation and Cell Death Induction by Ti-

vantinib and its Combination with Pemetrexed

Flow cytometry DNA analysis was performed to evaluate the effects of tivantinib, pemetrexed and their combination on cell-cycle distribution and to determine whether or not their cell-cycle alterations might provide any hint to optimize drug scheduling. As summarized in Table 1, tivantinib, pe-metrexed and their combination affected the cell cycle of all the MPM cells. In particular, tivantinib induced a G2/M ar-rest, with percentages of cells in this phase ranging from 4.4% to 12.5% in MSTO-211H control and treated cells, respectively, P<0.05. Similar perturbations of the cell cycle were observed after exposure to the drug combination. Con-versely, pemetrexed induced an S-phase arrest, increasing the fraction of cells in the G0/G1 in the H2452 and H2052 cells, and S-phase in the MSTO-211H and H28, supporting its cell-type specific activity on cell cycle perturbation.

Cell death (sub-G1 region) was significantly augmented after the treatments; this enhancement was more evident in the H28 cell line, with a 29.7% of cells in sub-G1 after the treatment with the combination. A total of death cells rang-ing between 4 and 15% were observed after tivantinib expo-sure; but in all the MPM cells the drug combination signifi-cantly increased the percentages of cell death with respect to both control cells and pemetrexed-treated cells.

Tivantinib Affects Tubulin Polymerization

Our findings on the modulation of the cell cycle as well as previous results in different cancer cell lines [24, 25] sug-

gest that tivantinib is a mitotic inhibitor. Since most antimi-totic agents impair cytoskeleton dynamics, we tested whether tivantinib could affect microtubule stability, using a vali-dated “whole cell” method that allows a rapid and quantita-tive analysis of tubulin polymerization levels within the cel-lular environment [31]. MSTO-211H and H2052 cells were incubated with IC50 concentrations of tivantinib and tubulin fluorescence was assessed by anti- -tubulin staining and flow cytometry. In this assay tivantinib induced a significant reduction in the fluorescence signal (Fig. 4), supporting its ability of disrupting microtubules in cells by abrogating mi-crotubule assembly. In contrast, compounds that induce cell death through other modes of action (i.e. pemetrexed) show no effect in this assay (data not shown).

Inhibition of cell Migration by Tivantinib and its Combi-

nation with Pemetrexed

The effect of tivantinib, pemetrexed and their combina-tion on MPM cells migratory behavior was investigated in MSTO-211H and H2052 using a scratch motility assay. As shown in Fig. (5A-B), tivantinib significantly reduced cell-migration in both MPM cells. This effect was already detect-able after 6 hours of exposure, with percentage of wound healing area of 9% in H2052 cells compared to 27% in un-treated cells (Fig. 5A). Similarly, in MSTO-211H cells we observed after 6 hours of exposure, that the percentage of the wound healing area was of 17% compared to 47% in un-treated cells (Fig. 5B). Furthermore, the percentages of mi-grated cells after 8-hour exposure in H2052 were approxi-mately 44%, 49%, 14% and 15% in untreated, pemetrexed,

Table 1. Cell-cycle modulation and cell death

Cells Treatment G0/G1

Phase (%)

S Phase

(%)

G2/M

Phase (%)

Death

Cells (%)

Control 74.6±9.9 21.1±7.6 4.4±1.3 2.3±0.7

Tivaninib 66.1±0.6 25.5±2.1 8.4±1.6 4.0±0.7

Pemetrexed 58.1±5.3 36.4±3.3 5.5±0.8 9.1±2.1

MSTO-211H

Pemetrexed+Tivaninib 53.3±5.9 39.8±9.2 6.9±0.1 17.2±5.9

Control 65.8±8.0 26.7±7.7 7.5±2.2 1.9±0.4

Tivatinib 56.4±6.6 22.2±8.4 21.4±1.0 14.7±5.5

Pemetrexed 72.5±6.1 24.0±2.8 3.5±0.9 7.5±1.7

H2452

Pemetrexed+Tivaninib 44.9±11.9 35.5±4.4 19.6±2.5 22.8±1.9

Control 61.7±9.6 34.9±6.9 3.4±0.74 1.6±0.7

Tivatinib 47.6±0.2 35.9±0.6 16.5±0.4 3.5±1.8

Pemetrexed 70.6±6.4 24.2±3.3 5.2±0.9 4.9±0.8

H2052

Pemetrexed+Tivaninib 34.2±3.8 22.3±6.47 43.5±3.4 13.5±5.0

Control 72.5±1.7 18.2±0.1 9.3±1.8 0.7±0.3

Tivatinib 34.3±4.9 33.0±1.26 32.7±6.7 15.8±5.3

Pemetrexed 45.0±7.8 45.5±4.1 9.5±1.5 5.5±1.1

H28

Pemetrexed+Tivaninib 38.8±2.9 47.2±3.7 14.0±2.4 29.7±7.2

Tivantinib & Pemetrexed in MPM Current Drug Targets, 2014, Vol. 15, No. 14 1335

Fig. (4). Tivantinib affects tubulin polymerization. Representative histograms from flow cytometry studies to assess tubulin fluorescence,

suggesting destabilization of the microtubules in both the (A) MSTO-211H and (B) H2052 cells.

Fig. (5). Effects of tivantinib, pemetrexed and their combination on MPM cells migration. Results of wound-healing assay in H2052 (A)

and MSTO-211H cells (B) exposed to tivantinib 10 μM, pemetrexed and their combination for 24 hours. The ability of the cells to migrate

into the wound area was assessed by comparing the pixels in the images taken at the beginning of the exposure (time 0), with those taken after

4, 6, 8, and 24 hours. Columns, mean values obtained from three independent duplicate experiments; bars, SEM. P<0.05, significantly differ-

ent from controls.

tivantinib and tivantinib/pemetrexed combination treated cells, respectively. Similar results were observed in the MSTO-211H cells. These data demonstrate that the drug combination was also able to significantly inhibit the migra-tion of MPM cells compared to both untreated and pe-metrexed-treated cells, both after 8 and 24 hours exposure (P<0.05).

DISCUSSION

The present study demonstrates that tivantinib has growth inhibitory effects in a panel of human MPM cell lines char-acterized by distinct molecular properties. Limited published preclinical research focusing on c-Met inhibition showed similar "cytotoxic activity for the experimental first-generation small-molecule inhibitor of c-Met SU11274, par-ticularly in the cell lines H513 and H2596, harboring the T1010I mutation, which exhibited the most dramatic reduc-tion of cell growth when compared with wild-type H28 and

nonmalignant MeT-5A cells”, as reported previously [15]. Similarly, the second-generation small-molecule inhibitor of c-Met, PHA-665752, exerted its inhibitory of cell growth activity only in two MPM cell lines that exhibited a c-Met/HGF autocrine loop, H2461 and JMN-1B [35], while only the combination of c-Met and EGFR inhibitors trig-gered stronger inhibition on cell proliferation and invasion in in vitro experiments on four primary MPM cell cultures and three established cell lines (MSTO-211H, NCI-H2373 and NCI-H290) [17]. These findings suggested that activation of multiple receptors TK is critical for cell proliferation and/or survival of MPM cells and that the specific inhibition of c-Met has limited impact on MPM.

However, in the present study we observed that sensitiv-ity to tivantinib in MPM cell lines felt within the range of IC50 values for other tumor cell lines, including "lung, pan-creas, breast, colon, gastric, brain, ovarian and thyroid cancer cells, independently of c-MET gene amplification and c-Met

1336 Current Drug Targets, 2014, Vol. 15, No. 14 Leon et al.

protein expression” as previously reported [24, 25, 36, 37]. Previous studies in different tumor types indeed demon-strated that the biological activity of tivantinib is not due solely to c-Met inhibition [24, 25], and showed that ti-vantinib is a microtubule-active agent. In agreement with these data, our results demonstrated, for the first time in MPM cells, that tivantinib is able to inhibit c-Met but acts also a mitotic inhibitor, which increases the percentages of cells in the G2/M phase and reduces the content of tubulin, resulting in abrogation of the microtubule assembly.

These preclinical findings are extremely important be-cause of their potential clinical implications, through the identification of additional predictive biomarkers for ti-vantinib in addition to c-Met. Moreover, recent bioinformat-ics analysis of microarray data from 53 MPM samples fol-lowed by pathway analysis revealed that the cytoskele-ton/spindle microtubules network was the second-most sig-nificantly affected network [26].

The analysis of compatibility with existing therapies rep-resents the basis for the clinical translation of novel drugs, and, to the best of our knowledge, this is the first study evaluating the pharmacological interaction of tivantinib with pemetrexed in MPM. Our results showed that tivantinib in-teracted synergistically with pemetrexed and increased the apoptotic fraction induced by pemetrexed treatment. These findings are in agreement with our previous studies showing enhanced apoptotic cell death after combined treatment of pemetrexed with other TKIs such as vandetanib or erlotinib in MPM and lung cancer cell lines [28, 38].

Previous studies on TKIs in malignant MPM cells have been performed using single drugs, such as gefitinib, er-lotinib, and canertinib [39]. However, combination targeting of kinase signaling pathways as well as combination between targeted and chemotherapeutic agents seem more effective than single agents in several MPM cells [40, 41]. These data fit with the concept that an additional inhibition may be re-quired to prevent that the inhibition of only one pathway by a TK antagonist might lead to optimal use of alternative sig-naling pathways. Indeed our findings show that the synergis-tic interaction between pemetrexed and tivantinib is medi-ated by several mechanisms, which enhanced the sensitivity to both drugs, and should be further evaluated as potential predictive biomarkers for the future clinical development of this or similar combinations.

Keeping with previous evidence in lung and pancreatic cancer cells the increase of pemetrexed activity in the com-bination with tivantinib may be the result of the modulation of cell cycle, with an increased number of cells in the S and G2/M phase, potentially facilitating the activity of the anti-metabolite pemetrexed in actively replicating cells [42, 43].

However, our findings show that the synergistic interac-tion of tivantinib with pemetrexed is also mediated by other mechanisms, which specifically enhanced the cytotoxic ac-tivity of pemetrexed, while reducing the aggressiveness of MPM cells. Indeed, tivantinib significantly reduced the ex-pression of TS, which has been significantly correlated with pemetrexed sensitivity of MPM both in the preclinical and in the clinical setting [28, 42, 44]. These data are in agreement with previous observations that not only antifolates, 5-FU, but also new targeted agents modulate TS [28, 45].

Finally, the present study demonstrated that tivantinib significantly reduced the migration of MPM cells, and this inhibitory effect was further enhanced in the cells treated with the tivantinib/pemetrexed combination. These data are in agreement with recent findings, showing that tivantinib, alone and in combination with and zoledronic acid, exhibited dose-dependent antimigratory and antimetastatic activity both in vitro and in vivo, inducing significant inhibition of metastatic growth of breast cancer cells in bone [46, 47]. These effects might be explained by the modulation of c-Met signaling, as already observed on seven MPM cell lines, where “HGF stimulated c-Met tyrosine phosphorylation, and this was associated with enhanced cell migration. Con-versely, HGF-dependent Met activation and migration were inhibited by NK4", as reported previously [48].

In conclusion, tivantinib emerged as a promising antican-cer agent against MPM, by attacking key mechanisms in-volved in cell cycle control, apoptosis and migration. Moreover, the favorable modulation of TS underlying the tivantinib/pemetrexed synergistic interaction makes this combination an excellent candidate to be tested in vivo, and may have implications for the rational clinical development of innovative regimens.

LIST OF ABBREVIATIONS

5-FU = 5 Fluorouracil

AbDil = Antibody Diluting Solution

ATCC = American Type Culture Collection

cDNA = Complementary DNA

CI = Combination index

c-Met = Hepatocyte Growth Factor Receptor

DNA = Deoxyribonucleic acid

EDTA = Ethylenediaminetetraacetic acid

EGFR = Epidermal growth factor receptor

ELISA = Enzyme-linked immunosorbent assay

Fa = Fraction affected

FAM = 6-carboxyfluorescein

FITC = Fluorescein isothiocyanate

FPGS = Folylpolyglutamate-sintase

HGF = Hepatocyte growth factor

IC50 = Inhibitory concentration 50%

MPM = Malignant pleural mesothelioma

mRNA = Messenger RNA

MTSB = Microtubule stabilizing buffer

NaBH4 = Sodium tetrahydridoborate

NK4 = Hepatocyte growth factor-antagonist inhibitor

OS = Overall survival

PBS = Phosphate buffered saline

PCR = Polymerase chain reaction

PET = Positron emission tomography

Tivantinib & Pemetrexed in MPM Current Drug Targets, 2014, Vol. 15, No. 14 1337

PI = Propidium iodide

RFC = Reduced folate carrier

RNA = Ribonucleic acid

RNAse = Ribonuclease

RPPH1 = Ribonuclease P RNA component H1 gene

RR = Response rate

RT-PCR = Reverse transcriptase quantitative polymerase chain reaction

SD = Standard deviation

SEM = Standard error of the mean

SRB = Sulforhodamine-B-assay

TKIs = Tyrosine kinase inhibitors

TS = Thymidylate synthase

TTP = Time-to-progression

CONFLICT OF INTEREST

The authors confirm that this article content has no con-flict of interest.

ACKNOWLEDGEMENTS

This work was supported by grants from Fondazione Humanitas (to E. Giovannetti and M. Gemelli), "The Law Offices of Peter G. Angelos Grant" from the Mesothelioma Applied Research Foundation (to LG Leon and E. Giovan-netti), FP7-REGPOT-2012-CT2012-31637-IMBRAIN (to LG Leon and E. Giovannetti); AIRC/Start-Up (to E. Giovan-netti).

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Received: July 15, 2014 Revised: August 20, 2014 Accepted: October 21, 2014